Mutant Alleles at the Brown Locus Encoding Tyrosinase‐Related Protein‐1 (TRP‐1) Affect Proliferation of Mouse Melanocytes in Culture *

Tyrosinase related protein‐1 (TRP‐1) is a melanocyte‐specific gene product involved in eumelanin synthesis. Mutation in the Tyrp1 gene is associated with brown pelage in mouse and oculocutaneous albinism Type 3 in humans (OCA3). It has been demonstrated that TRP‐1 expresses DHICA oxidase activity in the murine system. However, its actual function in the human system is still unclear. The study was designed to determine the effects of mutation at two Typr1 alleles, namely the Tyrp1^b (brown) and Tyrp1^b‐cj (cordovan) compared with wild type Tyrp1^B (black) on melanocyte function and melanin biosynthesis. The most significant finding was that both of the Tyrp1 mutations (i.e. brown expressing a point mutation and cordovan expressing decreased amount of TRP‐1 protein) resulted in attenuation of cell proliferation rates. Neither necrosis nor apoptosis was responsible for the observed decrease in cell proliferation rates of the brown and cordovan melanocytes. Ultrastructural evaluation by electron microscopic analysis revealed that both mutations in Tyrp1 affected melanosome maturation without affecting its structure. These observations demonstrate that mutation in Tyrp1 compromised tyrosinase activity within the organelle. DOPA histochemistry revealed differences in melanosomal stages between black and brown melanocytes but not between black and cordovan melanocytes. There were no significant differences in tyrosine hydroxylase activities of tyrosinase and TRP‐1 in wild type black, brown and cordovan melanocyte cell lysates. We conclude that mutations in Tyrp1 compromise cell proliferation and melanosomal maturation in mouse melanocyte cultures.     

Glucose metabolism down‐regulates the uptake of 6‐(N‐(7‐nitrobenz‐2‐oxa‐1,3‐diazol‐4‐yl)amino)‐2‐deoxyglucose (6‐NBDG) mediated by glucose transporter 1 isoform (GLUT1): theory and simulations using the symmetric four‐state carrier model

The non‐metabolizable fluorescent glucose analogue 6‐(N‐(7‐nitrobenz‐2‐oxa‐1,3‐diazol‐4‐yl)amino)‐2‐deoxyglucose (6‐NBDG) is increasingly used to study cellular transport of glucose. Intracellular accumulation of exogenously applied 6‐NBDG is assumed to reflect concurrent gradient‐driven glucose uptake by glucose transporters (GLUTs). Here, theoretical considerations are provided that put this assumption into question. In particular, depending on the microscopic parameters of the carrier proteins, theory proves that changes in glucose transport can be accompanied by opposite changes in flow of 6‐NBDG. Simulations were carried out applying the symmetric four‐state carrier model on the GLUT1 isoform, which is the only isoform whose kinetic parameters are presently available. Results show that cellular 6‐NBDG uptake decreases with increasing rate of glucose utilization under core‐model conditions, supported by literature, namely where the transporter is assumed to work in regime of slow reorientation of the free‐carrier compared with the ligand–carrier complex. To observe an increase of 6‐NBDG uptake with increasing rate of glucose utilization, and thus interpret 6‐NBDG increase as surrogate of glucose uptake, the transporter must be assumed to operate in regime of slow ligand–carrier binding, a condition that is currently not supported by literature. Our findings suggest that the interpretation of data obtained with NBDG derivatives is presently ambiguous and should be cautious because the underlying transport kinetics are not adequately established.     

Theaflavin‐3,3´‐digallate increases the antibacterial activity of β‐lactam antibiotics by inhibiting metallo‐β‐lactamase activity

Metallo‐β‐lactamases (MBLs) are some of the best known β‐lactamases produced by common Gram‐positive and Gram‐negative pathogens and are crucial factors in the rise of bacterial resistance against β‐lactam antibiotics. Although many types of β‐lactamase inhibitors have been successfully developed and used in clinical settings, no MBL inhibitors have been identified to date. Nitrocefin, checkerboard and time‐kill assays were used to examine the enzyme behaviour in vitro. Molecular docking calculation, molecular dynamics simulation, calculation of the binding free energy and ligand‐residue interaction decomposition were used for mechanistic research. The behaviour of the enzymes in vivo was investigated by a mouse infection experiment. We showed that theaflavin‐3,3´‐digallate (TFDG), a natural compound lacking antibacterial activities, can inhibit the hydrolysis of MBLs. In the checkerboard and time‐kill assays, we observed a synergistic effect of TFDG with β‐lactam antibiotics against methicillin‐resistant Staphylococcus aureus BAA1717. Molecular dynamics simulations were used to identify the mechanism of the inhibition of MBLs by TFDG, and we observed that the hydrolysis activity of the MBLs was restricted by the binding of TFDG to Gln242 and Ser369. Furthermore, the combination of TFDG with β‐lactam antibiotics showed effective protection in a mouse Staphylococcus aureus pneumonia model. These findings suggest that TFDG can effectively inhibit the hydrolysis activity of MBLs and enhance the antibacterial activity of β‐lactam antibiotics against pathogens in vitro and in vivo.     

γ‐glutamyl transpeptidase 1 specifically suppresses green‐light avoidance via GABAA receptors in Drosophila

Drosophila larvae innately show light avoidance behavior. Compared with robust blue‐light avoidance, larvae exhibit relatively weaker green‐light responses. In our previous screening for genes involved in larval light avoidance, compared with control w¹¹¹⁸ larvae, larvae with γ‐glutamyl transpeptidase 1 (Ggt‐1) knockdown or Ggt‐1 mutation were found to exhibit higher percentage of green‐light avoidance which was mediated by Rhodopsin6 (Rh6) photoreceptors. However, their responses to blue light did not change significantly. By adjusting the expression level of Ggt‐1 in different tissues, we found that Ggt‐1 in malpighian tubules was both necessary and sufficient for green‐light avoidance. Our results showed that glutamate levels were lower in Ggt‐1 null mutants compared with controls. Feeding Ggt‐1 null mutants glutamate can normalize green‐light avoidance, indicating that high glutamate concentrations suppressed larval green‐light avoidance. However, rather than directly, glutamate affected green‐light avoidance indirectly through GABA, the level of which was also lower in Ggt‐1 mutants compared with controls. Mutants in glutamate decarboxylase 1, which encodes GABA synthase, and knockdown lines of the GABA_A receptor, both exhibit elevated levels of green‐light avoidance. Thus, our results elucidate the neurobiological mechanisms mediating green‐light avoidance, which was inhibited in wild‐type larvae.

     

A lycopene β‐cyclase/lycopene ε‐cyclase/light‐harvesting complex‐fusion protein from the green alga Ostreococcus lucimarinus can be modified to produce α‐carotene and β‐carotene at different ratios

Biosynthesis of asymmetric carotenoids such as α‐carotene and lutein in plants and green algae involves the two enzymes lycopene β‐cyclase (LCYB) and lycopene ε‐cyclase (LCYE). The two cyclases are closely related and probably resulted from an ancient gene duplication. While in most plants investigated so far the two cyclases are encoded by separate genes, prasinophyte algae of the order Mamiellales contain a single gene encoding a fusion protein comprised of LCYB, LCYE and a C‐terminal light‐harvesting complex (LHC) domain. Here we show that the lycopene cyclase fusion protein from Ostreococcus lucimarinus catalyzed the simultaneous formation of α‐carotene and β‐carotene when heterologously expressed in Escherichia coli. The stoichiometry of the two products in E. coli could be altered by gradual truncation of the C‐terminus, suggesting that the LHC domain may be involved in modulating the relative activities of the two cyclase domains in the algae. Partial deletions of the linker region between the cyclase domains or replacement of one or both cyclase domains with the corresponding cyclases from the green alga Chlamydomonas reinhardtii resulted in pronounced shifts of the α‐carotene‐to‐β‐carotene ratio, indicating that both the relative activities of the cyclase domains and the overall structure of the fusion protein have a strong impact on the product stoichiometry. The possibility to tune the product ratio of the lycopene cyclase fusion protein from Mamiellales renders it useful for the biotechnological production of the asymmetric carotenoids α‐carotene or lutein in bacteria or fungi.     

Characterization of an ethanol‐tolerant 1,4‐β‐xylosidase produced by Pichia membranifaciens

Aims: The purification and biochemical properties of the 1,4‐β‐xylosidase of an oenological yeast were investigated. Methods and Results: An ethanol‐tolerant 1,4‐β‐xylosidase was purified from cultures of a strain of Pichia membranifaciens grown on xylan at 28°C. The enzyme was purified by sequential chromatography on DEAE cellulose and Sephadex G‐100. The relative molecular mass of the enzyme was determined to be 50 kDa by SDS‐PAGE. The activity of 1,4‐β‐xylosidase was optimum at pH 6·0 and at 35°C. The activity had a K_m of 0·48 ± 0·06 mmol l^?1 and a V_max of 7·4 ± 0·1 μmol min^?1 mg^?1 protein for p‐nitrophenyl‐β‐d ‐xylopyranoside. Conclusions: The enzyme characteristics (pH and thermal stability, low inhibition rate by glucose and ethanol tolerance) make this enzyme a good candidate to be used in enzymatic production of xylose and improvement of hemicellulose saccharification for production of bioethanol. Significance and Impact of the Study: This study may be useful for assessing the ability of the 1,4‐β‐xylosidase from P. membranifaciens to be used in the bioethanol production process.     

2,6‐Dimethoxy‐1,4‐Benzoquinone Enhances Resistance Against the Rice Blast Fungus Magnaporthe oryzae

We investigated the effect of 2,6‐dimethoxy‐1,4‐benzoquinone (DMBQ) on induced resistance to Magnaporthe oryzae in rice. DMBQ concentrations greater than 50 μg/ml inhibited spore germination and appressorium formation in M. oryzae. When rice leaves pretreated with 10 μg/ml DMBQ, which did not show antifungal activity against spore germination and appressorium formation of M. oryzae, were inoculated with M. oryzae spores 5 days after DMBQ pretreatment, blast lesion formation was inhibited compared with control leaves pretreated with distilled water. In addition, infection‐inhibiting activity against M. oryzae was significantly enhanced in rice leaf sheaths pretreated with 10 μg/ml DMBQ. H₂O₂ generation was observed in rice leaves pretreated with DMBQ, and PAL, POX, CHS and PR10a were significantly expressed in these leaves. These results suggested that DMBQ can protect rice from blast disease caused by M. oryzae.     

Functional analysis of endo‐1,4‐β‐glucanases in response to Botrytis cinerea and Pseudomonas syringae reveals their involvement in plant–pathogen interactions

Plant cell wall modification is a critical component in stress responses. Endo‐1,4‐β‐glucanases (EGs) take part in cell wall editing processes, e.g. elongation, ripening and abscission. Here we studied the infection response of Solanum lycopersicum and Arabidopsis thaliana with impaired EGs. Transgenic TomCel1 and TomCel2 tomato antisense plants challenged with Pseudomonas syringae showed higher susceptibility, callose priming and increased jasmonic acid pathway marker gene expression. These two EGs could be resistance factors and may act as negative regulators of callose deposition, probably by interfering with the defence‐signalling network. A study of a set of Arabidopsis EG T‐DNA insertion mutants challenged with P. syringae and Botrytis cinerea revealed that the lack of other EGs interferes with infection phenotype, callose deposition, expression of signalling pathway marker genes and hormonal balance. We conclude that a lack of EGs could alter plant response to pathogens by modifying the properties of the cell wall and/or interfering with signalling pathways, contributing to generate the appropriate signalling outcomes. Analysis of microarray data demonstrates that EGs are differentially expressed upon many different plant–pathogen challenges, hormone treatments and many abiotic stresses. We found some Arabidopsis EG mutants with increased tolerance to osmotic and salt stress. Our results show that impairing EGs can alter plant–pathogen interactions and may contribute to appropriate signalling outcomes in many different biotic and abiotic plant stress responses.     

A novel Fe(II)/α‐ketoglutarate‐dependent dioxygenase from Burkholderia ambifaria has β‐hydroxylating activity of N‐succinyl l‐leucine

An Fe(II)/α‐ketoglutarate‐dependent dioxygenase, SadA, was obtained from Burkholderia ambifaria AMMD and heterologously expressed in Escherichia coli. Purified recombinant SadA had catalytic activity towards several N‐substituted l‐amino acids, which was especially strong with N‐succinyl l‐leucine. With the NMR and LC‐MS analysis, SadA converted N‐succinyl l‐leucine into N‐succinyl l‐threo‐β‐hydroxyleucine with >99% diastereoselectivity. SadA is the first enzyme catalysing β‐hydroxylation of aliphatic amino acid‐related substances and a potent biocatalyst for the preparation of optically active β‐hydroxy amino acids.     

Specificity of presenilin‐1‐ and presenilin‐2‐dependent γ‐secretases towards substrate processing

The two presenilin‐1 (PS1) and presenilin‐2 (PS2) homologs are the catalytic core of the γ‐secretase complex, which has a major role in cell fate decision and Alzheimer's disease (AD) progression. Understanding the precise contribution of PS1‐ and PS2‐dependent γ‐secretases to the production of β‐amyloid peptide (Aβ) from amyloid precursor protein (APP) remains an important challenge to design molecules efficiently modulating Aβ release without affecting the processing of other γ‐secretase substrates. To that end, we studied PS1‐ and PS2‐dependent substrate processing in murine cells lacking presenilins (PSs) (PS1KO, PS2KO or PS1‐PS2 double‐KO noted PSdKO) or stably re‐expressing human PS1 or PS2 in an endogenous PS‐null (PSdKO) background. We characterized the processing of APP and Notch on both endogenous and exogenous substrates, and we investigated the effect of pharmacological inhibitors targeting the PSs activity (DAPT and L‐685,458). We found that murine PS1 γ‐secretase plays a predominant role in APP and Notch processing when compared to murine PS2 γ‐secretase. The inhibitors blocked more efficiently murine PS2‐ than murine PS1‐dependent processing. Human PSs, especially human PS1, expression in a PS‐null background efficiently restored APP and Notch processing. Strikingly, and contrary to the results obtained on murine PSs, pharmacological inhibitors appear to preferentially target human PS1‐ than human PS2‐dependent γ‐secretase activity.