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.     

The two enantiospecific dichlorprop/α-ketoglutarate-dioxygenases from Delftia acidovorans MC1 – protein and sequence data of RdpA and SdpA

Two α-ketoglutarate-dependent dioxygenases carrying enantiospecific activity for the etherolytic cleavage of racemic phenoxypropionate herbicides [(RS)-2-(2,4-dichlorophenoxy)propionate and (RS)-2-(4-chloro-2-methylphenoxy)propionate] from Delftia acidovorans MC1 were characterized with respect to protein and sequence data. The (S)-phenoxypropionate/α-ketoglutarate-dioxygenase (SdpA) appeared as a monomeric enzyme with a molecular weight of 32 kDa in the presence of SDS. N-terminal sequences revealed relationship to α-ketoglutarate-dependent taurine dioxygenase (TauD) and to 2,4-dichlorophenoxyacetate/α-ketoglutarate-dioxygenase (TfdA). The (R)-phenoxypropionate/α-ketoglutarate-dioxygenase (RdpA) referred to 36 kDa in the presence of SDS and to 108 kDa under native conditions. Internal sequences of fragments obtained after digestion made evident relationship to TfdA and TauD. Two-dimensional electrophoretic separation resulted in the resolution of up to 3 individual spots with almost identical molecular weights but different isoelectric points with both RdpA and SdpA. The structural differences of these isoenzyme forms are not yet clear.     

A Theoretical Study on the Metabolic Requirements Resulting from α-Ketoglutarate-Dependent Cleavage of Phenoxyalkanoates

The etherolytic cleavage of phenoxyalkanoic acids in various bacteria is catalyzed by an α-ketoglutarate-dependent dioxygenase. In this reaction, the electron acceptor is oxidatively decarboxylated to succinate, whereas the proper substrate is cleaved by forming the oxidized alkanoic acid and the phenolic intermediate. The necessity of regenerating α-ketoglutarate and the consequences for the overall metabolism were investigated in a theoretical study. It was found that the dioxygenase mechanism is accompanied by a significant loss of carbon amounting to up to 62.5% in the assimilatory branch, thus defining the upper limit of carbon conversion efficiency. This loss in carbon is almost compensated for in comparison to a monooxygenase-catalyzed initial step when the dissimilatory efforts of the entire metabolism are included: the yield coefficients become similar. The α-ketoglutarate-dependent dioxygenase mechanism has more drastic consequences for microorganisms which are restricted in their metabolism to the first step of phenoxyalkanoate degradation by excreting the phenolic intermediate as a dead-end product. In the case of phenoxyacetate derivatives, the cleavage reaction would quickly cease due to the exhaustion of α-ketoglutarate and no growth would be possible. With the cleavage products of phenoxypropionate and phenoxybutyrate herbicides, i.e., pyruvate and succinate(semialdehyde), respectively, as the possible products, the regeneration of α-ketoglutarate will be guaranteed for stoichiometric reasons. However, the maintenance of the cleavage reaction ought to be restricted due to physiological factors owing to the involvement of other metabolic reactions in the pool of metabolites. These effects are discussed in terms of a putative recalcitrance of these compounds.     

Characterization of Two Novel α-Glucosidases from Bifidobacterium breve UCC2003

Two α-glucosidase-encoding genes (agl1 and agl2) from Bifidobacterium breve UCC2003 were identified and characterized. Based on their similarity to characterized carbohydrate hydrolases, the Agl1 and Agl2 enzymes are both assigned to a subgroup of the glycosyl hydrolase family 13, the α-1,6-glucosidases (EC 3.2.1.10). Recombinant Agl1 and Agl2 into which a His₁₂ sequence was incorporated (Agl1_His and Agl2_His, respectively) exhibited hydrolytic activity towards panose, isomaltose, isomaltotriose, and four sucrose isomers—palatinose, trehalulose, turanose, and maltulose—while also degrading trehalose and, to a lesser extent, nigerose. The preferred substrates for both enzymes were panose, isomaltose, and trehalulose. Furthermore, the pH and temperature optima for both enzymes were determined, showing that Agl1_His exhibits higher thermo and pH optima than Agl2_His. The two purified α-1,6-glucosidases were also shown to have transglycosylation activity, synthesizing oligosaccharides from palatinose, trehalulose, trehalose, panose, and isomaltotriose.     

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.     

Enantioselective Uptake and Degradation of the Chiral Herbicide Dichlorprop [(RS)-2-(2,4-Dichlorophenoxy)propanoic acid] by Sphingomonas herbicidovorans MH

Sphingomonas herbicidovorans MH was able to completely degrade both enantiomers of the chiral herbicide dichlorprop [(RS)-2-(2,4-dichlorophenoxy)propanoic acid], with preferential degradation of the (S) enantiomer over the (R) enantiomer. These results are in agreement with the recently reported enantioselective degradation of mecoprop [(RS)-2-(4-chloro-2-methylphenoxy)propanoic acid] by this bacterium (C. Zipper, K. Nickel, W. Angst, and H.-P. E. Kohler, Appl. Environ. Microbiol. 62:4318–4322, 1996). Uptake of (R)-dichlorprop, (S)-dichlorprop, and 2,4-D (2,4-dichlorophenoxyacetic acid) was inducible. Initial uptake rates of cells grown on the respective substrate showed substrate saturation kinetics with apparent affinity constants (K_t) of 108, 93, and 117 μM and maximal velocities (V_max) of 19, 10, and 21 nmol min⁻¹ mg of protein⁻¹ for (R)-dichlorprop, (S)-dichlorprop, and 2,4-D, respectively. Transport of (R)-dichlorprop, (S)-dichlorprop, and 2,4-D was completely inhibited by various uncouplers and by nigericin but was only marginally inhibited by valinomycin and by the ATPase inhibitor N,N′-dicyclohexylcarbodiimine. Experiments on the substrate specificity of the putative transport systems revealed that (R)-dichlorprop uptake was inhibited by (R)-mecoprop but not by (S)-mecoprop, (S)-dichlorprop, or 2,4-D. On the other hand, the (S)-dichlorprop transport was inhibited by (S)-mecoprop but not by (R)-mecoprop, (R)-dichlorprop, or 2,4-D. These results provide evidence that the first step in the degradation of dichlorprop, mecoprop, and 2,4-D by S. herbicidovorans is active transport and that three inducible, proton gradient-driven uptake systems exist: one for (R)-dichlorprop and (R)-mecoprop, another for (S)-dichlorprop and (S)-mecoprop, and a third for 2,4-D.     

Enantioselective Transformation of α-Hexachlorocyclohexane by the Dehydrochlorinases LinA1 and LinA2 from the Soil Bacterium Sphingomonas paucimobilis B90A

Sphingomonas paucimobilis B90A contains two variants, LinA1 and LinA2, of a dehydrochlorinase that catalyzes the first and second steps in the metabolism of hexachlorocyclohexanes (R. Kumari, S. Subudhi, M. Suar, G. Dhingra, V. Raina, C. Dogra, S. Lal, J. R. van der Meer, C. Holliger, and R. Lal, Appl. Environ. Microbiol. 68:6021-6028, 2002). On the amino acid level, LinA1 and LinA2 were 88% identical to each other, and LinA2 was 100% identical to LinA of S. paucimobilis UT26. Incubation of chiral α-hexachlorocyclohexane (α-HCH) with Escherichia coli BL21 expressing functional LinA1 and LinA2 S-glutathione transferase fusion proteins showed that LinA1 preferentially converted the (+) enantiomer, whereas LinA2 preferred the (−) enantiomer. Concurrent formation and subsequent dissipation of β-pentachlorocyclohexene enantiomers was also observed in these experiments, indicating that there was enantioselective formation and/or dissipation of these enantiomers. LinA1 preferentially formed (3S,4S,5R,6R)-1,3,4,5,6-pentachlorocyclohexene, and LinA2 preferentially formed (3R,4R,5S,6S)-1,3,4,5,6-pentachlorocyclohexene. Because enantioselectivity was not observed in incubations with whole cells of S. paucimobilis B90A, we concluded that LinA1 and LinA2 are equally active in this organism. The enantioselective transformation of chiral α-HCH by LinA1 and LinA2 provides the first evidence of the molecular basis for the changed enantiomer composition of α-HCH in many natural environments. Enantioselective degradation may be one of the key processes determining enantiomer composition, especially when strains that contain only one of the linA genes, such as S. paucimobilis UT26, prevail.     

Purification and Characterization of l-Methionine γ-Lyase from Brevibacterium linens BL2

l-Methionine γ-lyase (EC 4.4.1.11) was purified to homogeneity from Brevibacterium linens BL2, a coryneform bacterium which has been used successfully as an adjunct bacterium to improve the flavor of Cheddar cheese. The enzyme catalyzes the α,γ elimination of methionine to produce methanethiol, α-ketobutyrate, and ammonia. It is a pyridoxal phosphate-dependent enzyme, with a native molecular mass of approximately 170 kDa, consisting of four identical subunits of 43 kDa each. The purified enzyme had optimum activity at pH 7.5 and was stable at pHs ranging from 6.0 to 8.0 for 24 h. The pure enzyme had its highest activity at 25°C but was active between 5 and 50°C. Activity was inhibited by carbonyl reagents, completely inactivated by dl-propargylglycine, and unaffected by metal-chelating agents. The pure enzyme had catalytic properties similar to those of l-methionine γ-lyase from Pseudomonas putida. Its K_m for the catalysis of methionine was 6.12 mM, and its maximum rate of catalysis was 7.0 μmol min⁻¹ mg⁻¹. The enzyme was active under salt and pH conditions found in ripening Cheddar cheese but susceptible to degradation by intracellular proteases.

Methanethiol is associated with desirable Cheddar-type sulfur notes in good-quality Cheddar cheese (2, 27). The mechanism for the production of methanethiol in cheese is unknown, but it is linked to the catabolism of methionine (1, 15). l-Methionine γ-lyase (EC 4.4.1.11; MGL), also known as methionase, l-methionine γ-demethiolase, and l-methionine methanethiollyase (deaminating), is a pyridoxal phosphate (PLP)-dependent enzyme that catalyzes the direct conversion of l-methionine to α-ketobutyrate, methanethiol, and ammonia by an α,γ-elimination reaction (26). It does not catalyze the conversion of d enantiomers (24–26). MGL in Pseudomonas putida is a multifunctional enzyme system since it catalyzes the α,γ- and α,β-elimination reactions of methionine and its derivatives (24). In addition, the enzyme also catalyzes the β-replacement reactions of sulfur amino acids (24). Since its discovery in Escherichia coli and Proteus vulgaris by Onitake (19), this enzyme has been found in various bacteria and is regarded as a key enzyme in the bacterial metabolism of methionine. However, this enzyme has not been purified to homogeneity from any food-grade microorganisms.MGL is widely distributed in bacteria, especially in pseudomonads, and is induced by the addition of l-methionine to the culture medium (9, 28). The enzyme has been purified from Pseudomonas putida (25), Aeromonas sp. (26), Clostridium sporogenes (11), and Trichomonas vaginalis (16) and partially purified from and characterized for Brevibacterium linens NCDO 739 (4).B. linens is a nonmotile, non-spore-forming, non-acid-fast, gram-positive coryneform bacterium normally found on the surfaces of Limburger and other Trappist-type cheeses. This organism tolerates salt concentrations ranging between 8 and 20% and is capable of growing in a broad pH range from 5.5 to 9.5, with an optimum pH of 7.0 (20). In Trappist-type cheeses, brevibacteria depend on Saccharomyces cerevisiae to metabolize lactate, which increases the pH of the curd, as well as to produce growth factors that are important for their growth (20). Interest in B. linens has focused around its ability to produce an extracellular protease, which has recently been isolated (21), and its ability to produce high levels of methanethiol (3, 9, 10, 22).B. linens produces various sulfur compounds, including methanethiol, that are thought to be important in Cheddar-like flavor and aroma (3, 9, 10, 22). Ferchichi et al. (9) suggested that MGL is responsible for the methanethiol-producing capability of B. linens but did not provide definitive evidence. Weimer et al. (28) proposed that B. linens BL2 is responsible for Cheddar-type flavor development in low-fat cheese, but again conclusive evidence was lacking. In this study, MGL was purified to homogeneity from B. linens BL2 and its physical and chemical properties were examined.     

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.     

Isolation and Characterization of Novel Psychrophilic, Neutrophilic, Fe-Oxidizing, Chemolithoautotrophic α- and γ-Proteobacteria from the Deep Sea

We report the isolation and physiological characterization of novel, psychrophilic, iron-oxidizing bacteria (FeOB) from low-temperature weathering habitats in the vicinity of the Juan de Fuca deep-sea hydrothermal area. The FeOB were cultured from the surfaces of weathered rock and metalliferous sediments. They are capable of growth on a variety of natural and synthetic solid rock and mineral substrates, such as pyrite (FeS₂), basalt glass (~10 wt% FeO), and siderite (FeCO₃), as their sole energy source, as well as numerous aqueous Fe substrates. Growth temperature characteristics correspond to the in situ environmental conditions of sample origin; the FeOB grow optimally at 3 to 10°C and at generation times ranging from 57 to 74 h. They are obligate chemolithoautotrophs and grow optimally under microaerobic conditions in the presence of an oxygen gradient or anaerobically in the presence of nitrate. None of the strains are capable of using any organic or alternate inorganic substrates tested. The bacteria are phylogenetically diverse and have no close Fe-oxidizing or autotrophic relatives represented in pure culture. One group of isolates are γ-Proteobacteria most closely related to the heterotrophic bacterium Marinobacter aquaeolei (87 to 94% sequence similarity). A second group of isolates are α-Proteobacteria most closely related to the deep-sea heterotrophic bacterium Hyphomonas jannaschiana (81 to 89% sequence similarity). This study provides further evidence for the evolutionarily widespread capacity for Fe oxidation among bacteria and suggests that FeOB may play an unrecognized geomicrobiological role in rock weathering in the deep sea.     

Lytic Effects of Staphylococcal α-Toxin and δ-Hemolysin

Several preparations of staphylococcal alpha-toxin and delta-lysin were studied in order to compare hemolytic activity with capacity to lyse bacterial protoplasts. delta-Lysin in relatively low concentration lysed protoplasts of Sarcina lutea, protoplasts of Streptococcus faecalis, and spheroplasts of Escherichia coli. Lysis of bacterial protoplasts by preparations of alpha-toxin appeared to be due to contamination of the preparations with delta-lysin. Data comparing the protoplast-lysing activity of various lytic agents are presented.     

Production, Purification, and Characterization of β-(1-4)-Endoxylanase of Streptomyces roseiscleroticus

Twelve species of Streptomyces that formerly belonged to the genus Chainia were screened for the production of xylanase and cellulase. One species, Streptomyces roseiscleroticus (Chainia rosea) NRRL B-11019, produced up to 16.2 IU of xylanase per ml in 48 h. A xylanase from S. roseiscleroticus was purified and characterized. The enzyme was a debranching β-(1-4)-endoxylanase showing high activity on xylan but essentially no activity against acid-swollen (Walseth) cellulose. It had a very low apparent molecular weight of 5,500 by native gel filtration, but its denatured molecular weight was 22,600 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It had an isoelectric point of 9.5. The pH and temperature optima for hydrolysis of arabinoxylan were 6.5 to 7.0 and 60°C, respectively, and more than 75% of the optimum enzyme activity was retained at pH 8.0. The xylanase had a K_m of 7.9 mg/ml and an apparent V_max of 305 μmol · min^-1 · mg of protein^-1. The hydrolysis rate was linear for xylan concentrations of less than 4 mg/ml, but significant inhibition was observed at xylan concentrations of more than 10 mg/ml. The predominant products of arabinoxylan hydrolysis included arabinose, xylobiose, and xylotriose.     

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.     

Purification and Properties of Staphylococcal δ-Hemolysin I. Production of δ-Hemolysin

Concentrated preparations of staphylococcal delta-hemolysin were obtained by growing selected hemolytic colonies from the 146P strain of Staphylococcus aureus on dialysis membranes laid over Brain Liver Heart agar plates at 37 C for 20 hr under 10% CO(2) and harvesting the growth from five such membranes in 1.0 ml of deionized distilled water. Incubation in a humid environment facilitated this harvesting procedure. Incubation longer than 40 hr or incubation under CO(2) higher than 10 to 20% gave lower yields of delta-lysin. Addition of a sugar, fermented by the organisms, resulted in lower yields of delta-hemolysin. Agar, although separated from the growing cells by the dialysis membrane, did potentiate delta-hemolysin production. Addition of 0.1% agar to the inoculum further enhanced this potentiation. delta-Hemolysin produced in broth or semisolid cultures was excessively diluted with the media. Dialysis membranes prevented this dilution and thus yielded concentrated preparations of delta-hemolysin.     

Construction and Characterization of Salmonella typhimurium Strains That Accumulate and Excrete α- and β- Isopropylmalate

Two Salmonella typhimurium strains, which could be used as sources for the leucine biosynthetic intermediates α- and β-isopropylmalate were constructed by a series of P22-mediated transductions. One strain, JK527 [flr-19 leuA2010 Δ(leuD-ara)798 fol-162], accumulated and excreted α-isopropylmalate, whereas the second strain, JK553 (flr-19 leuA2010 leuB698), accumulated and excreted α- and β-isopropylmalate. The yield of α-isopropylmalate isolated from the culture medium of JK527 was more than five times the amount obtained from a comparable volume of medium in which Neurospora crassa strain FLR₉₂-1-216 (normally used as the source for α- and β-isopropylmalate) was grown. Not only was the yield greater, but S. typhimurium strains are much easier to handle and grow to saturation much faster than N. crassa strains. The combination of the two regulatory mutations flr-19, which results in constitutive expression of the leucine operon, and leuA2010, which renders the first leucine-specific biosynthetic enzyme insensitive to feedback inhibition by leucine, generated limitations in the production of valine and pantothenic acid. The efficient, irreversible, and unregulated conversion of α-ketoisovaleric acid into α-isopropylmalate (α-isopropylmalate synthetase K_m for α-ketoisovaleric acid, 6 × 10⁻⁵ M) severely restricted the amount of α-ketoisovaleric acid available for conversion into valine and pantothenic acid (ketopantoate hydroxymethyltransferase K_m for α-ketoisovaleric acid, 1.1 × 10⁻³ M; transaminase B K_m for α-ketoisovaleric acid, 2 × 10⁻³ M).     

Characterization of α1(IV) Collagen Mutations in Caenorhabditis elegans and the Effects of α1 and α2(IV) Mutations on Type IV Collagen Distribution

Type IV collagen is a major component of basement membranes. We have characterized 11 mutations in emb-9, the α1(IV) collagen gene of Caenorhabditis elegans, that result in a spectrum of phenotypes. Five are substitutions of glycines in the Gly-X-Y domain and cause semidominant, temperature-sensitive lethality at the twofold stage of embryogenesis. One is a glycine substitution that causes recessive, non–temperature-sensitive larval lethality. Three putative null alleles, two nonsense mutations and a deletion, all cause recessive, non–temperature-sensitive lethality at the threefold stage of embryogenesis. The less severe null phenotype indicates that glycine substitution containing mutant chains dominantly interfere with the function of other molecules. The emb-9 null mutants do not stain with anti–EMB-9 antisera and show intracellular accumulation of the α2(IV) chain, LET-2, indicating that LET-2 assembly and/or secretion requires EMB-9. Glycine substitutions in either EMB-9 or LET-2 cause intracellular accumulation of both chains. The degree of intracellular accumulation differs depending on the allele and temperature and correlates with the severity of the phenotype. Temperature sensitivity appears to result from reduced assembly/secretion of type IV collagen, not defective function in the basement membrane. Because the dominant interference of glycine substitution mutations is maximal when type IV collagen secretion is totally blocked, this interference appears to occur intracellularly, rather than in the basement membrane. We suggest that the nature of dominant interference caused by mutations in type IV collagen is different than that caused by mutations in fibrillar collagens.     

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.     

Differential Subcellular Localization of Protein Phosphatase-1 α, γ1, and δ Isoforms during Both Interphase and Mitosis in Mammalian Cells

Protein phosphatase-1 (PP-1) is involved in the regulation of numerous metabolic processes in mammalian cells. The major isoforms of PP-1, α, γ1, and δ, have nearly identical catalytic domains, but they vary in sequence at their extreme NH₂ and COOH termini. With specific antibodies raised against the unique COOH-terminal sequence of each isoform, we find that the three PP-1 isoforms are each expressed in all mammalian cells tested, but that they localize within these cells in a strikingly distinct and characteristic manner. Each isoform is present both within the cytoplasm and in the nucleus during interphase. Within the nucleus, PP-1 α associates with the nuclear matrix, PP-1 γ1 concentrates in nucleoli in association with RNA, and PP-1 δ localizes to nonnucleolar whole chromatin. During mitosis, PP-1 α is localized to the centrosome, PP-1 γ1 is associated with microtubules of the mitotic spindle, and PP-1 δ strongly associates with chromosomes. We conclude that PP-1 isoforms are targeted to strikingly distinct and independent sites in the cell, permitting unique and independent roles for each of the isoforms in regulating discrete cellular processes.     

Bile Salt 3α- and 12α-Hydroxysteroid Dehydrogenases from Eubacterium lentum and Related Organisms

Thirty-two strains of Eubacterium lentum and phenotypically similar anaerobic gram-positive bacilli were screened for intracellular bile salt 3α- and 12α-hydroxysteroid dehydrogenase (HSDHase) activities. These organisms were categorized into four groups: (A) those containing 12α-HSDHase only (10 strains), (B) those containing 3α- and 12α-HSDHase (13 strains), (C) those containing 3α-HSDHase only (2 strains), and (D) those devoid of any measurable HSDHase activity (7 strains). Of the respective four groups, 9/10, 13/13, 0/2, and 0/7 were like the neotype strain of E. lentum (ATCC 25559) in that they produced H₂S in a triple sugar iron agar butt, reduced nitrate to nitrite, and weakly decomposed hydrogen peroxide. The other strains were variable for nitrate reduction and activity on hydrogen peroxide, but all the organisms in the first three categories (with one exception) were H₂S producers (triple sugar iron agar butt) and all (with one exception) were designated E. lentum, whereas the organisms of category B were non-H₂S producers (triple sugar iron agar butt). Five of these seven were not stimulated by arginine and are designated “phenotypically similar organisms.” Thin-layer chromatography of extracted spent bacterial medium of four representative strains from each group grown in the presence of cholate revealed the presence of (A) 12-oxo product, (B) 12-oxo and 3-oxo products, (C) 3-oxo product, and (D) the absence of any of these products. The 12α-HSDHase of category B organisms was unstable unless 10⁻³ M dithioerythritol was added to the buffer. With the exception of 3 out of 32 strains, there was a positive correlation between the production of measurable amounts of 12α-HSDHase and H₂S production. Growth curves and the effect of arginine on growth and the production of 3α- and 12α-HSDHase were examined in representative strains of categories A, B, and C. Both enzymes were shown to bind onto a nicotinamide adenine dinucleotide-Sepharose column and could be eluted by high-ionic-strength buffer, resulting in approximately 25-fold and 18-fold purification, respectively. Molecular weight estimations by Sephadex G-200 gave values of 205,000 and 125,000 for the 3α- and 12α-HSDHase, respectively. Purified 12α-HSDHase was investigated with respect to pH requirement, substrate specificity, and enzyme kinetics.     

Haloperoxidases: Enzymatic Synthesis of α,β-Halohydrins from Gaseous Alkenes

The enzymatic synthesis of α,β-halohydrins from gaseous alkenes is described. The enzymatic reaction required an alkene, a halide ion, dilute hydrogen peroxide, and a haloperoxidase enzyme. A wide range of gaseous alkenes were suitable for this reaction, including those containing isolated, conjugated, and cumulative carbon-carbon double bonds. Chlorohydrins, bromohydrins, and iodohydrins could be formed. The combining of this enzymatic synthesis with a previously described enzymatic synthesis of epoxides from α,β-halohydrins provides an alternate pathway, other than the well-known enzymatic direct epoxidation pathway, from alkene to an epoxide.