Laccase‐catalysed polymeric dye synthesis from plant‐derived phenols for potential application in hair dyeing: Enzymatic colourations driven by homo‐ or hetero‐polymer synthesis

Laccase efficiently catalyses polymerization of phenolic compounds. However, knowledge on applications of polymers synthesized in this manner remains scarce. Here, the potential of laccase-catalysed polymerization of natural phenols to form products useful in hair dyeing was investigated. All 15 tested phenols yielded coloured products after laccase treatment and colour diversity was attained by using mixtures of two phenolic monomers. After exploring colour differentiation pattern of 120 different reactions with statistical regression analysis, three monomer combinations, namely gallic acid and syringic acid, catechin and catechol, and ferulic acid and syringic acid, giving rise to brown, black, and red materials, respectively, were further characterized because such colours are commercially important for grey hair dyeing. Selected polymers could strongly absorb visible light and their hydrodynamic sizes ranged from 100 to 400 nm. Analyses of enzyme kinetic constants, liquid chromatography and electrospray ionization-mass spectrometry (ESI-MS) coupled with collision-induced dissociation MS/MS indicate that both monomers in reactions involving catechin and catechol, and ferulic acid and syringic acid, are coloured by heteropolymer synthesis, but the gallic acid/syringic acid combination is based on homopolymer mixture formation. Comparison of colour parameters from these three reactions with those of corresponding artificial homopolymer mixtures also supported the idea that laccase may catalyse either hetero- or homo-polymer synthesis. We finally used selected materials to dye grey hair. Each material coloured hair appropriately and the dyeing showed excellent resistance to conventional shampooing. Our study indicates that laccase-catalysed polymerization of natural phenols is applicable to the development of new cosmetic pigments.     

Monoclonal antibodies to rhamnogalacturonan I backbone

Monoclonal antibodies were raised against rhamnogalacturonan I backbone, a pectin domain, using Arabidopsis thaliana seed mucilage-derived rhamnogalacturonan I oligosaccharides—BSA conjugates. Two monoclonal antibodies, designated INRA-RU1 and INRA-RU2, selected for further characterization, were specific for the backbone of rhamnogalacturonan I, displaying no binding activity against the other pectin domains i.e. homogalacturonans, galactans or arabinans. A range of oligosaccharides was prepared by enzymatic digestion of rhamnogalacturonan I isolated from Arabidopsis thaliana seed mucilage and from sugar beet pectin, purified by low-pressure chromatography and characterized by high-performance anion-exchange chromatography and mass spectrometry. These rhamnogalacturonan I oligomers were used to characterize the binding site of the two monoclonal antibodies by competitive inhibition. Both INRA-RU1 and INRA-RU2 showed maximal binding to the [→2)-α-l-rhamnosep-(1→4)-α-d-galacturonic acid p-(1→]₇ structural motif but differed in their minimum binding requirement. INRA-RU2 required at least two disaccharide (rhamnose–galacturonic acid) repeats for the antibody to bind, while INRA-RU1 required a minimum of six disaccharide repeats. Furthermore, the binding capacity of INRA-RU1 decreased steeply as the number of disaccharide repeats go beyond seven. Each of these antibodies reacted with hairy regions isolated from sugar beet pectin. Immunofluorescence microscopy indicated that both antibodies can be readily used to detect rhamnogalacturonan I epitopes in various cell wall samples.     

Roles of alternative splicing in the functional properties of inner ear-specific KCNQ4 channels

The function of the KCNQ4 channel in the auditory setting is crucial to hearing, underpinned by the finding that mutations of the channel result in an autosomal dominant form of nonsyndromic progressive high frequency hearing loss. The precise function of KCNQ4 in the inner ear has not been established. However, recently we demonstrated that there is differential expression among four splice variants of KCNQ4 (KCNQ4_v1-v4) along the tonotopic axis of the cochlea. Alternative splicing specifies the outcome of functional channels by modifying the amino acid sequences within the C terminus at a site designated as the membrane proximal region. We show that variations within the C terminus of splice variants produce profound differences in the voltage-dependent phenotype and functional expression of the channel. KCNQ4_v4 lacks exons 9-11, resulting in deletion of 54 amino acid residues adjacent to the S6 domain compared with KCNQ4_v1. Consequently, the voltage-dependent activation of KCNQ4_v4 is shifted leftward by approximately 20 mV, and the number of functional channels is increased severalfold compared with KCNQ4_v1. The properties of KCNQ4_v2 and KCNQ4_v3 fall between KCNQ4_v1 and KCNQ4_v4. Because of variations in the calmodulin binding domains of the splice variants, the channels are differentially modulated by calmodulin. Co-expression of these splice variants yielded current magnitudes suggesting that the channels are composed of heterotetramers. Indeed, a dominant negative mutant of KCNQ4_v1 cripples the currents of the entire KCNQ4 channel family. Furthermore, the dominant negative KCNQ4 mutant stifles the activity of KCNQ2-5, raising the possibility of a global disruption of KCNQ channel activity and the ensuing auditory phenotype.     

Genomewide scan for linkage reveals evidence of several susceptibility loci for alopecia areata

Alopecia areata (AA) is a genetically determined, immune-mediated disorder of the hair follicle that affects 1%-2% of the U.S. population. It is defined by a spectrum of severity that ranges from patchy localized hair loss on the scalp to the complete absence of hair everywhere on the body. In an effort to define the genetic basis of AA, we performed a genomewide search for linkage in 20 families with AA consisting of 102 affected and 118 unaffected individuals from the United States and Israel. Our analysis revealed evidence of at least four susceptibility loci on chromosomes 6, 10, 16 and 18, by use of several different statistical approaches. Fine-mapping analysis with additional families yielded a maximum multipoint LOD score of 3.93 on chromosome 18, a two-point affected sib pair (ASP) LOD score of 3.11 on chromosome 16, several ASP LOD scores >2.00 on chromosome 6q, and a haplotype-based relative risk LOD of 2.00 on chromosome 6p (in the major histocompatibility complex locus). Our findings confirm previous studies of association of the human leukocyte antigen locus with human AA, as well as the C3H-HeJ mouse model for AA. Interestingly, the major loci on chromosomes 16 and 18 coincide with loci for psoriasis reported elsewhere. These results suggest that these regions may harbor gene(s) involved in a number of different skin and hair disorders.     

Inhibitors of HCV NS5B polymerase: synthesis and structure-activity relationships of unsymmetrical 1-hydroxy-4,4-dialkyl-3-oxo-3,4-dihydronaphthalene benzothiadiazine derivatives

Substituted 1-hydroxy-4,4-dialkyl-3-oxo-3,4-dihydronaphthalene benzothiadiazine derivatives were investigated as inhibitors of genotype 1 HCV polymerase. Structure-activity relationship patterns for this class of compounds are discussed. It was found that the saturated alkane dialkyl units provided the most active analogs.     

Comparative molecular characterization of ADSS1 and ADSS2 genes in pig (Sus scrofa)

Adenylosuccinate synthetase (ADSS) catalyzes the key step of AMP synthesis. Vertebrates have two isozymes of ADSS, which are named ADSS1 and ADSS2, respectively. In this study, we cloned porcine ADSS1 and ADSS2 genes and comparatively analyzed their sequence, chromosome mapping, mRNA distribution and subcellular localization. According to our results, the ADSS1 gene was predominantly expressed in the striated muscle tissues, while ADSS2 gene distributed widely in all the tissues detected. Additionally, ADSS1 gene was up-regulated significantly along with porcine muscle growth, and ADSS2 gene expression was more constant during the muscle development. Porcine ADSS1 gene was assigned to SSC7q and the linked marker was SSC12B09, ADSS2 gene was mapped on SSC10p and the linked marker was SW497, and porcine ADSS2 protein was subcellular localized in mitochondria. Moreover, we found that one single nucleotide polymorphism (SNP, T/C(70)) in the ninth intron of ADSS2 gene was significantly associated with average daily gain trait (ADG, P<0.05) and loin muscle area trait (P<0.05).     

The role of endogenous bioregulators in formation of cardiogenic reflex effects on circulation

The possible role of endogenous endothelium-derived bioactive substances in organization of cardiogenic depressor reflexes under cardiac receptor stimulation (by veratrine and bradykinin) was investigated in acute experiments on anesthetized rats. The results have shown that endothelium-derived bioactive substances take part in forming of the cardiogenic depressor reflex humoral components of nervous response or nervous modulators. These data contribute to understanding of the role of endogenous endothelium-derived bioactive substances (prostacyclin) and different NOS isoforms in mechanisms of depressor reflex development and species differences in their involvement in reflex vasomotor reactions.     

Litterfall Production Along Successional and Altitudinal Gradients of Subtropical Monsoon Evergreen Broadleaved Forests in Guangdong, China

Evaluation of litterfall production is important for understanding nutrient cycling, forest growth, successional pathways, and interactions with environmental variables in forest ecosystems. Litterfall was intensively studied during the period of 1982–2001 in two subtropical monsoon vegetation gradients in the Dinghushan Biosphere Reserve, Guangdong Province, China. The two gradients include: (1) a successional gradient composed of pine forest (PF), mixed pine and broadleaved forest (MF) and monsoon evergreen broadleaved forest (BF), and (2) an altitudinal gradient composed of Baiyunci ravine rain forest (BRF), Qingyunci ravine rain forest (QRF), BF and mountainous evergreen broadleaved forest (MMF). Mean annual litterfall production was 356, 861 and 849 g m⁻² for PF, MF and BF of the successional gradient, and 1016, 1061, 849 and 489 g m⁻² for BRF, QRF, BF and MMF of the altitudinal gradient, respectively. As expected, mean annual litterfall of the pioneer forest PF was the lowest, but rapidly increased over the observation period while those in other forests were relatively stable, confirming that forest litterfall production is closely related to successional stages and growth patterns. Leaf proportions of total litterfall in PF, MF, BF, BRF, QRF and MMF were 76.4%, 68.4%, 56.8%, 55.7%, 57.6% and 69.2%, respectively, which were consistent with the results from studies in other evergreen broadleaved forests. Our analysis on litterfall monthly distributions indicated that litterfall production was much higher during the period of April to September compared to other months for all studied forest types. Although there were significant impacts of some climate variables (maximum and effective temperatures) on litterfall production in some of the studied forests, the mechanisms of how climate factors (temperature and rainfall) interactively affect litterfall await further study.     

Generation of shrnas from randomized oligonucleotides

Suppression of gene expression by small interfering RNA (siRNA) has proved to be a gene-specific and cost effective alternative to other gene suppression technologies. Short hairpin RNAs (shRNAs) generated from the vector-based expression are believed to be processed into functional siRNAs in vivo, leading to gene silencing. Since an shRNA library carries a large pool of potential siRNAs, such a library makes it possible to knock down gene expression at the genome wide scale. Although much of research has been focused on generating shRNA libraries from either individually made gene specific sequences or cDNA libraries, there is no report on constructing randomized shRNA libraries, which could provide a good alternative to these existing libraries. We have developed a method of constructing shRNAs from randomized oligonucleotides. Through this method, one can generate a partially or fully randomized shRNA library for various functional analyses. We validated this procedure by constructing a p53-specific shRNA. Western blot revealed that the p53-shRNA successfully suppressed expression of the endogenous p53 in MCF-7 cells. We then made a partially randomized shRNA library. Sequencing of 15 randomly picked cloned confirmed the randomness of the library. Therefore, the library can be used for various functional assays, such as target validation when a suitable screening or selection method is available.