β-Glucosidase of Aspergillus aculeatus

Two kinds of cytochrome c oxidase were partially purified from iron-grown T. ferrooxidans. The first type (cytochrome c oxidase I) was easily solubilized without a detergent and had a pH-optimum at 3.0. The other (cytochrome c oxidase II) which was bound tightly to the cell membrane and solubilized with sodium dodecyl sulfate had a pH-optimum at 5.2. Each type was heat-sensitive and inhibited by cyanide and azide. Since the pH level of the bacterial iron oxidizing activity corresponded closely with the pH of cytochrome c oxidase I but not cytochrome c oxidase II, it was proposed that the former may play an important role in the iron oxidizing system.     

Synthesis of 7β-hydroxy-epiandrosterone

The synthesis of 7β-hydroxy-epiandrosterone (6) possessing strong anti-inflammatory properties was achieved starting from 3β-acetoxy-17,17-(ethylenedioxy)-5-androsten (1). This approach involved as a main step an allylic oxidation of the C-7 followed by two reduction reactions of the double bond and of the carbonyl group. This stereoselective synthesis in 5 steps gave 7β-hydroxy-epiandrosterone in 63% overall yield.     

Synthesis of α- and γ-l-Glutamyl Dipeptides of l-β-Phenyl-β-Alanine

α- and γ-l-Glutamyl dipeptides of l-β-phenyl-β-alanine are synthesized for the first time from l-glutamic acid and l-β-phenyl-β-alanine. In addition, the preparations and the properties of new intermediates, that is, l-β-phenyl-β-alanine benzylester p-toluenesulfonate and the N-carbobenzyloxy-α- and γ-dipeptide benzylesters, are described. Further proof of the structure previously proposed for the naturally occurring peptide is obtained by a critical comparison of the isolated and synthetic materials by various physical and chemical methods.     

Structure-activity relationships of 9β-estrogens

The 9β isomers of estradiol-17β, estradiol-17α, estrone and 17-ethinylestradiol-17β were synthesized and compared with their 9α-counterparts in the rat uterine cytosol estrogen receptor, utero-tropic, and gonadotropin release inhibition assays. Except for 17-ethinyl-9β-estradiol-17β which was as active as its 9α isomer in the uterotropic assay, none of the 9β estrogens exhibited any biological activity which was equal to or greater than their 9α counterparts. For examples, 9β-estradiol-17β was $\text{1}\text{10}$ as active as estradiol-17β, and 9β-estrone was $\text{1}\text{4}$ as active as estrone in the uterotropic assay.     

β-Adrenergic receptors of frog erythrocytes

Following persistent stimulation of -adrenergic receptors of frog erythrocytes with (–)-isoproterenol, the cyclic adenosine 3,5-monophosphate-dependent protein kinase (cAMP-dependent protein kinase) (EC 2.7.1.37) was activated for several hours. This activation outlasted the duration of the increase of cAMP content. Following a persistant stimulation of -adrenergic receptors with isoproterenol, the phosphorylation of selective membrane proteins was increased. This increase in phosphorylation lasted longer than 4 hr but less than 12 hr. Between 2 and 4 hr after receptor stimulation the loss of -adrenergic receptor form plasma membrane was maximal, and the phosphorylation of two membrane proteins characterized by molecular weights of 60,000 and 38,000 daltons was selectively enhanced. In addition we found that isolated erythrocytes are capable of synthesizing RNA and polypeptides and that incubation with (–)-isoproterenol induces a longterm delayed increase of the synthesis of erythrocyte proteins. This increase in the synthesis of proteins appears to require new RNA synthesis. Thus the possibility can be entertained that this delayed increase in protein synthesis participates in the new synthesis of receptor and is operative in the termination of -adrenergic receptor subsensitivity elicited by a persistent stimulation with (–)-isoproterenol.     

β-D-Phosphogalactoside Galactohydrolase of Lactic Streptococci

beta-D-Phosphogalactoside galactohydrolase (beta-Pgal) was examined in a number of lactic streptococci by use of the chromogenic substrate o-nitrophenyl-beta-D-galactopyranoside-6-phosphate. Specific activity of beta-Pgal ranged from 0.563 units/mg of protein in Streptococcus lactis UN, to 0.120 in S. diacetilactics 18-16. Essentially no beta-D-galactoside galactohydrolase (beta-gal) was found in these organisms when o-nitrophenyl-beta-D-galactopyranoside served as the chromogenic substrate. S. lactis 7962 was the one exception found. This organism contained rather high levels of beta-gal, and very little beta-Pgal could be detected. beta-Pgal activity was examined in streptococci that differed widely in both their proteolytic ability and rates of lactic acid production during growth in milk. Differences in proteolytic ability did not influence beta-Pgal synthesis; also, the rate of lactic acid production was independent of the level of beta-Pgal present in the cell, since the rate of lactic acid production could be increased approximately fourfold without changing the amount of beta-Pgal present in the cell. Various carbohydrates were tested as potential inducers of the enzyme. Although galactose, either as the free sugar or combined with glucose in lactose, was the only inducer, noninducing sugars such as mannose or glucose showed some ability to cause fluctuations in the basal level of beta-Pgal. Cells growing in mannose or glucose exhibited about 30% of the maximal enzyme levels found in cells growing in lactose or galactose. No gratuitous inducers were found.     

Photophysical properties ofβ-homo-tyrosine derivatives

Summary The observed monoexponential fluorescence decay of N-acetyl--homo-tyrosine methylamide (Ac-Hty-NHMe) (I) and N-acetyl-(O-methyl)--homo-tyrosine methylamide (Ac-Hty(OMe)-NHMe) (II) is supporting the rotamer population theory, according to which rotamers are responsible for heterogeneity of the fluorescence decay of N-acetyl-tyrosine amide or tyrosine incorporated within a peptide chain, in general.     

β-Glucuronidase-coupled assays of glucuronoyl esterases

Glucuronoyl esterases (GEs) are microbial enzymes with potential to cleave the ester bonds between lignin alcohols and xylan-bound 4-O-methyl-d-glucuronic acid in plant cell walls. This activity renders GEs attractive research targets for biotechnological applications. One of the factors impeding the progress in GE research is the lack of suitable substrates. In this work, we report a facile preparation of methyl esters of chromogenic 4-nitrophenyl and 5-bromo-4-chloro-3-indolyl β-D-glucuronides for qualitative and quantitative GE assay coupled with β-glucuronidase as the auxiliary enzyme. The indolyl derivative affording a blue indigo-type product is suitable for rapid and sensitive assay of GE in commercial preparations as well as for high throughput screening of microorganisms and genomic and metagenomic libraries.     

Microbial 1β-Hydroxylation of Digitoxigenin

l-Deoxy-l-l-proIino-d-fructose was isolated from flue-cured tobacco leaves in crystalline form. The structure was confirmed by comparison with synthetic compound.     

β-Arrestin-Dependent Deactivation of Mouse Melanopsin

In mammals, the expression of the unusual visual pigment, melanopsin, is restricted to a small subset of intrinsically photosensitive retinal ganglion cells (ipRGCs), whose signaling regulate numerous non-visual functions including sleep, circadian photoentrainment and pupillary constriction. IpRGCs exhibit attenuated electrical responses following sequential and prolonged light exposures indicative of an adaptational response. The molecular mechanisms underlying deactivation and adaptation in ipRGCs however, have yet to be fully elucidated. The role of melanopsin phosphorylation and β-arrestin binding in this adaptive process is suggested by the phosphorylation-dependent reduction of melanopsin signaling in vitro and the ubiquitous expression of β-arrestin in the retina. These observations, along with the conspicuous absence of visual arrestin in ipRGCs, suggest that a β-arrestin terminates melanopsin signaling. Here, we describe a light- and phosphorylation- dependent reduction in melanopsin signaling mediated by both β-arrestin 1 and β-arrestin 2. Using an in vitro calcium imaging assay, we demonstrate that increasing the cellular concentration of β-arrestin 1 and β-arrestin 2 significantly increases the rate of deactivation of light-activated melanopsin in HEK293 cells. Furthermore, we show that this response is dependent on melanopsin carboxyl-tail phosphorylation. Crosslinking and co-immunoprecipitation experiments confirm β-arrestin 1 and β-arrestin 2 bind to melanopsin in a light- and phosphorylation- dependent manner. These data are further supported by proximity ligation assays (PLA), which demonstrate a melanopsin/β-arrestin interaction in HEK293 cells and ipRGCs. Together, these results suggest that melanopsin signaling is terminated in a light- and phosphorylation-dependent manner through the binding of a β-arrestin within the retina.     

Self-Assembly of Aβ40, Aβ42 and Aβ43 Peptides in Aqueous Mixtures of Fluorinated Alcohols

Fluorinated alcohols such as hexafluoroisopropanol (HFIP) and trifluoroethanol (TFE) have the ability to promote α-helix and β-hairpin structure in proteins and peptides. HFIP has been used extensively to dissolve various amyloidogenic proteins and peptides including Aβ, in order to ensure their monomeric status. In this paper, we have investigated the self-assembly of Aβ40, Aβ42, and Aβ43 in aqueous mixtures of fluorinated alcohols from freshly dissolved stock solutions in HFIP. We have observed that formation of fibrillar and non-fibrillar structures are dependent on the solvent composition. Peptides form fibrils with ease when reconstituted in deionized water from freshly dissolved HFIP stocks. In aqueous mixtures of fluorinated alcohols, either predominant fibrillar structures or clustered aggregates were observed. Aqueous mixtures of 20% HFIP are more favourable for Aβ fibril formation as compared to 20% TFE. When Aβ40, Aβ42, and Aβ43 stocks in HFIP are diluted in 50% aqueous mixtures in phosphate buffer or deionized water followed by slow evaporation of HFIP, Aβ peptides form fibrils in phosphate buffer and deionized water. The clustered structures could be off-pathway aggregates. Aβ40, Aβ42, and Aβ43 showed significant α-helical content in freshly dissolved HFIP stocks. The α-helical conformational intermediate in Aβ40, Aβ42, and Aβ43 could favour the formation of both fibrillar and non-fibrillar aggregates depending on solvent conditions and rate of α-helical to β-sheet transition.     

Discovery of a novel covalent non-β-lactam inhibitor of the metallo-β-lactamase NDM-1

The inhibition of metallo-β-lactamases (MBL) can prevent the hydrolysis of β-lactam antibiotics and hence is a promising strategy for the treatment of antibiotic resistant infections. In this study, we present a novel reversible covalent inhibitor of the clinically relevant MBL New Delhi metallo-β-lactamase 1 (NDM-1). Electrospray ionization-mass spectrometry (ESI-MS) and single site directed mutagenesis were used to show that the inhibitor forms a covalent bond with Lys224 in the active site of NDM-1. The inhibitor was further characterized using an enzyme inhibition assay, a surface plasmon resonance (SPR) based biosensor assay and covalent docking. The determined inhibition constant (K_I¹) was 580 nM and the inhibition constant for the initial complex (K_I) was 76 μM. To our knowledge, this inhibitor is the first example for a reversible covalent non-β-lactam inhibitor targeting NDM-1 and a promising starting point for the design of potent covalent inhibitors.     

Development of inclusion complex of brinzolamide with hydroxypropyl-β-cyclodextrin

Glaucoma is an accumulative optic neuropathy resulted from increasing intraocular pressure. Brinzolamide (BRZ) is a kind of carbonic anhydrase inhibitors for glaucoma treatment. In this study, brinzolamide-hydroxypropyl-β-cyclodextrin (BRZ-HP-β-CD) inclusion complex was prepared by solvent evaporation method to improve the solubility of BRZ and enhance the therapeutic effect of BRZ. The formation of the inclusion complex was confirmed by Fourier transform infrared spectroscopy, differential scanning calorimeter and nuclear magnetic resonance spectroscopy. The solubility of BRZ increased about 10-fold after the formation of the BRZ-HP-β-CD inclusion complex. The in vitro corneal accumulative permeability of the inclusion complex increased 2.91-fold compared to the commercial available formulation (AZOPT^®). In addition, BRZ-HP-β-CD inclusion complex (0.5% BRZ) had an equivalent efficiency of lowering intraocular pressure with AZOPT^® (1% BRZ) in vivo. These results identified the BRZ-HP-β-CD inclusion complex might have a promising future as a novel formulation of BRZ for glaucoma treatment.     

Investigation of inclusion complex of cilnidipine with hydroxypropyl-β-cyclodextrin

The objective of this study was to improve the water-solubility and photostability of cilnidipine by complexing it with hydroxypropyl-β-cyclodextrin (HP-β-CD or HP-beta-CD). The interactions of cilnidipine and HP-β-CD were characterized by ultra violet-visible (UV/VIS) spectroscopy, differential scanning calorimetry (DSC), powder X-ray diffraction (PXRD), Fourier transformation-infrared (FT-IR) spectroscopy and (1)H nuclear magnetic resonance ((1)H NMR) spectroscopy to verify the formation of cilnidipine-HP-β-CD complex inclusion. Moreover, the binding sites in the HP-β-CD structure were also tracked through (1)H NMR spectroscopy analysis. All the characterization information proved the formation of cilnidipine-HP-β-CD inclusion complex, and the results demonstrated the superiority of the inclusion complex in dissolution rates and photostability; in addition, the apparent solubility of cilnidipine was increased more than 10,000-fold in the presence of HP-β-CD. The stability constant (1:1) was found to be 50,116M(-1), suggesting a high tendency of the drug to enter the HP-β-CD cavity. These results identified the cilnidipine-HP-β-CD inclusion complex as an effective new approach to design a novel formulation for pharmaceutical application.     

Persistence of TGF-β1 induction of increased fibroblast contractility

Fibroblast contraction of collagen gels is regarded as a model of wound contraction. Transforming growth factor (TGF)-beta added to such gels can augment contraction consistent with its suggested role as a mediator of fibrotic repair. Since fibroblasts isolated from fibrotic tissues have been suggested to express a "fibrotic phenotype," we hypothesized that TGF-beta exposure may lead to a persistent increase in fibroblasts' contractility. To evaluate this question, confluent human fetal lung fibroblasts were treated with serum-free Dulbecco modified Eagle medium (DMEM), with or without 100 pM [corrected] TGF-beta1, TGF-beta2, or TGF-beta3 for 48 h. Fibroblasts were then trypsinized and cast into gels composed of native type I collagen isolated from rat tail tendons. After 20 min for gelation, the gels were released and maintained in serum-free DMEM. TGF-beta-pretreated fibroblasts caused significantly more rapid gel contraction (52.5+/-0.6, 50.9+/-0.2, and 50.3+/-0.5% by TGF-beta1, -beta2, and -beta3 pretreated fibroblasts, respectively) than control fibroblasts (74.0+/-0.3%, P < 0.01). This effect is concentration dependent (50-200 nM), and all three isoforms had equal activity. The effect of TGF-beta1, however, persisted for only a short period of time following the removal of TGF-beta, and was lost with sequential passage. These observations suggest that the persistent increase in collagen-gel contractility, mediated by fibroblasts from fibrotic tissues, would not appear to be solely due to previous exposure of these cells to TGF-beta.     

Effects of propionate on accumulation of poly(β-hydroxy-butyrate-co-β-hydroxyvalerate) and excretion of pyruvate in Alcaligenes eutrophus

Summary Effects of propionate on the accumulation of poly(-hydroxybutyrate-co--hydroxyvalerate) and the excretion of pyruvate in Alcaligenes eutrophus were investigated at various concentrations of glucose and propionate. As propionate concentration increased, an enhancement in pyruvate excretion was observed along with a decrease in the yield of the copolymer. At the same concentration of propionate, hydroxyvalerate content of the copolymer was reduced from 26 to 15 mol % with increase of the initial glucose concentration.