Annotation and De Novo Sequence Characterization of Extracellular β-Fructofuranosidase from <Emphasis Type="Italic">Penicillium chrysogenum</Emphasis> Strain HKF42

The genome of a fungal strain Penicillium chrysogenum strain HKF42, which can grow on 20% sucrose has been annotated for 7595 protein coding sequences. On mining of CAZymes, we could annotate a β-fructofuranosidase gene responsible for fructo-oligosaccharides (FOS) synthesis which is a known prebiotic. The enzyme activity was demonstrated and validated with the generation of FOS as kestose and nystose.     

Energetics of gating at the apo–acetylcholine receptor transmitter binding site

Acetylcholine receptor channels switch between conformations that have a low versus high affinity for the transmitter and conductance for ions (R↔R*; gating). The forward isomerization, which begins at the transmitter binding sites and propagates ∼50 Å to the narrow region of the pore, occurs by approximately the same sequence of molecular events with or without agonists present at the binding sites. To pinpoint the forces that govern the R versus R* agonist affinity ratio, we measured single-channel activation parameters for apo-receptors having combinations of mutations of 10 transmitter binding site residues in the α (Y93, G147, W149, G153, Y190, C192, and Y198), ε (W55 and P121), or δ (W57) subunit. Gating energy changes were largest for the tryptophan residues. The αW149 energy changes were coupled with those of the other aromatic amino acids. Mutating the aromatic residues to Phe reduces the R/R* equilibrium dissociation constant ratio, with αY190 and αW149 being the most sensitive positions. Most of the mutations eliminated long-lived spontaneous openings. The results provide a foundation for understanding how ligands trigger protein conformational change.     

Prognostic biomarkers for survival in mucosal melanoma

Mucosal melanoma (MM) is a rare subtype of melanoma with an aggressive clinical course. In cutaneous melanoma (CM), the absence of pigmentation and presence of NRAS/KRAS mutations are biomarkers indicating an aggressive clinical course with shorter overall survival. Similar data for MM are missing. We present the real-world outcome data in a cohort of genotyped MM patients and assessed the prognostic relevance of pigmentation- and NRAS/KRAS mutation status. We correlated pathological reports and clinical data with overall survival of patients with MM. Furthermore, we performed clinically integrated molecular genotyping and analyzed real world treatment regimens for covariates associated with clinical outcome. We identified 39 patients with available clinical and molecular data. Patients with amelanotic MM had a significantly shorter overall survival (p = .003). In addition, the presence of a NRAS or KRAS mutation was significantly associated with poor overall survival (NRAS or KRAS p = .024). Currently, it is unknown if the same prognostic relevance for the lack of pigmentation and RAS mutations in CM, exists in MM. Here we analyzed a cohort of MM for outcome measures and determined that two known prognostic biomarkers for CM are in fact novel prognosticators for MM.     

Tibolone and its delta-4, 7α-methyl norethisterone metabolite are reversible inhibitors of human aromatase

Tibolone is used for the treatment of climacteric symptoms and osteoporosis in menopausal women. After ingestion, it is rapidly converted to a number of metabolites including 3α- and 3β-hydroxy derivatives and the delta-4, 7α-methylnorethisterone (7α-MeNET) metabolite, which is rapidly cleared from circulation. Tibolone and some of its metabolites act in a tissue-selective manner to inhibit steroid sulphatase (STS) and 17β-hydroxysteroid dehydrogenase Type 1 (17β-HSD1) activities but also stimulate steroid sulphotransferase and 17β-HSD2 activities. In the present study we have examined whether the ability of tibolone and its 7α-MeNET metabolites to regulate the activities of enzymes involved in oestrogen formation or inactivation extends to another key enzyme involved in oestrogen synthesis, the aromatase, which converts androstenedione to oestrone. Using JEG-3 choriocarcinoma cells, which have a high level of aromatase activity, tibolone and 7α-MeNET, but not the 3α- or 3β-hydroxy metabolites, were found to inhibit aromatase activity in intact cells and also lysates prepared from these cells (up to 61% inhibition at 10 μM). An investigation into the nature of aromatase inhibition by these compounds revealed that they inhibit aromatase activity by a reversible mechanism. Tibolone and 7α-MeNET also inhibited aromatase activity in MCF-7 breast cancer cells, which have a much lower level of aromatase activity than JEG-3 cells. It is concluded that, in addition to inhibiting STS and 17β-HSD1, tibolone and 7α-MeNET may exert some of their tissue-selective effects in regulating oestrogen synthesis by also inhibiting aromatase activity.     

Mining the metagenome of activated biomass of an industrial wastewater treatment plant by a novel method

Metagenomic libraries herald the era of magnifying the microbial world, tapping into the vast metabolic potential of uncultivated microbes, and enhancing the rate of discovery of novel genes and pathways. In this paper, we describe a method that facilitates the extraction of metagenomic DNA from activated sludge of an industrial wastewater treatment plant and its use in mining the metagenome via library construction. The efficiency of this method was demonstrated by the large representation of the bacterial genome in the constructed metagenomic libraries and by the functional clones obtained. The BAC library represented 95.6 times the bacterial genome, while, the pUC library represented 41.7 times the bacterial genome. Twelve clones in the BAC library demonstrated lipolytic activity, while four clones demonstrated dioxygenase activity. Four clones in pUC library tested positive for cellulase activity. This method, using FTA cards, not only can be used for library construction, but can also store the metagenome at room temperature.     

Effect of growth temperature,induction, and molecular chaperones on the solubilization of over-expressed cellobiose phosphorylase from <Emphasis Type="Italic">Cellvibrio Gilvus</Emphasis> under <Emphasis Type="Italic">in vivo</Emphasis> conditions

In vivo folding of many proteins can be facilitated by growth temperature, extent of induction, and molecular chaperones, which prevent over-expressed protein from being trapped into insoluble inclusion bodies. In the present report, we describe the role of molecular chaperones and growth temperature on the solubilization of overexpressed Cellobiose Phosphorylase (CBP) in Escherichia coli. The growth of host at low temperature enhanced enzyme in soluble fraction. Similarly, induction of target gene at low level of IPTG also yielded higher enzyme in soluble fraction. The synergistic effect of low temperature and induction on the prevention of inclusion bodies was also evident from our results. In addition, co-expression of the target gene with two types of molecular chaperones (GroESL and KODHsp) was also attempted. However, none of these chaperones enhanced the solubilization under in vivo conditions. Nevertheless, effective role of low growth temperature coupled with low level of induction appeared to be an attractive feature for producing recombinant protein.     

Discovery and validation of serum protein changes in type 1 diabetes patients using high throughput two dimensional liquid chromatography-mass spectrometry and immunoassays

Type 1 diabetes (T1D) is expected to cause significant changes in the serum proteome; however, few studies have systematically assessed the proteomic profile change associated with the disease. In this study, a semiquantitative spectral counting-based two dimensional liquid chromatography mass spectrometry platform was used to analyze serum samples from T1D patients and controls. In this discovery phase, significant differences were found for 21 serum proteins implicated in inflammation, oxidation, metabolic regulation, and autoimmunity. To assess the validity of these findings, six candidate proteins including adiponectin, insulin-like growth factor binding protein 2, serum amyloid protein A, C-reactive protein, myeloperoxidase, and transforming growth factor beta induced were selected for subsequent immune assays for 1139 T1D patients and 848 controls. A series of statistical analyses using cases and controls matched for age, sex, and genetic risk confirmed that T1D patients have significantly higher serum levels for four of the six proteins: adiponectin (odds ratio (OR) = 1.95, p = 10(-27)), insulin-like growth factor binding protein 2 (OR = 2.02, p < 10(-20)), C-reactive protein (OR = 1.13, p = 0.007), serum amyloid protein A (OR = 1.51, p < 10(-16)); whereas the serum levels were significantly lower in patients than controls for the two other proteins: transforming growth factor beta induced (OR = 0.74, p < 10(-5)) and myeloperoxidase (OR = 0.51, p < 10(-41)). Compared with subjects in the bottom quartile, subjects in the top quartile for adiponectin (OR = 6.29, p < 10(-37)), insulin-like growth factor binding protein 2 (OR = 7.95, p < 10(-46)), C-reactive protein (OR = 1.38, p = 0.025), serum amyloid protein A (OR = 3.36, p < 10(-16)) had the highest risk of T1D, whereas subjects in the top quartile of transforming growth factor beta induced (OR = 0.41, p < 10(-11)) and myeloperoxidase (OR = 0.10, p < 10(-43)) had the lowest risk of T1D. These findings provided valuable information on the proteomic changes in the sera of T1D patients.     

Lactic dehydrogenase activity and cellular localization of several dehydrogenases in Neurospora and Allomyces

Summary Lactic dehydrogenase (LDH) activity has been cytochemically localized and its activity measured in wild-type and mutant strains of Neurospora crassa and male and female hybrids of Allomyces.In all strains, less intracellular staining is found, by oxidative assay of lactic dehydrogenase, ethanol dehydrogenase and a few other dehydrogenases, in the hyphal tips than in the older regions of the hyphae.The extractible activity of LDH, assayed reductively in the soluble fraction, is much greater in Allomyces than Neurospora. In Allomyces the least activity is found in the female differentiated strain. The male differentiated strain and especially the vegetative cultures of both strains have much more activity. In Neurospora, conidiating cultures have unexpectedly more activity than vegetative cultures. The crisp mutant which forms increased numbers of conidia has more activity than the wild-type which, in turn, has more activity than the aconidial fluffy mutant.     

Responses of Cajanus cajan and rhizospheric N-cycling communities to bioinoculants

Background and aims

Bioinoculants are commonly used for enhancing crop productivity but little information is available on their effect on key microbial communities such as those involved in the cycling of nitrogen, a major plant nutrient. Here we developed a formulation combining different bioinoculants (Bacillus megaterium, Pseudomonas fluorescens and Trichoderma harzianum) and examined their effects on both Cajanus cajan growth and N-cycling microorganisms.

Methods

Seven bioinoculant combinations were evaluated in pots under field conditions, and their effects on plant growth were measured using various biometric parameters. The abundances of the total bacterial and crenarchaeal communities along with those involved in N-cycling were monitored by qPCR at vegetative, pre-flowering, flowering and maturity stages of the crop.

Results

A significant increase in growth of C. cajan was observed when treated with mixture of three bioinoculants with dry biomass and grain yield increase by 330?% and 238?%, respectively. The combination of three bioinoculants also increased the abundance of nitrogen fixers and denitrifiers towards the flowering and maturity stages.

Conclusions

The consortium of three bioinoculants increased plant growth and grain yield of C. cajan. These bioinoculants also had a positive effect on the abundance of several N-cycling microbial communities stressing the importance of understanding non-target effects of bioinoculants together with their impact on plant growth.     

Interaction of Hermansky-Pudlak Syndrome genes in the regulation of lysosome-related organelles

Hermansky-Pudlak Syndrome (HPS) is a genetically heterogeneous disease caused by abnormalities in the synthesis and/or trafficking of lysosome-related organelles (LROs) including melanosomes, lamellar bodies of lung type II cells and platelet dense granules. At least 15 genes cause HPS in mice, with a significant number specifying novel subunits of protein complexes termed BLOCs (Biogenesis of Lysosome-related Organelles Complexes). To ascertain whether BLOC complexes functionally interact in vivo, mutant mice doubly or triply deficient in protein subunits of the various BLOC complexes and/or the AP-3 adaptor complex were constructed and tested for viability and for abnormalities of melanosomes, lung lamellar bodies and lysosomes. All mutants, including those deficient in all three BLOC complexes, were viable though the breeding efficiencies of multiple mutants involving AP-3 were severely compromised. Interactions of BLOC protein complexes with each other and with AP-3 to affect most LROs were apparent. However, these interactions were tissue and organelle dependent. These studies document novel biological interactions of BLOC and AP-3 complexes in the biosynthesis of LROs and assess the role(s) of HPS protein complexes in general health and physiology in mammals. Double and triple mutant HPS mice provide unique and practical experimental advantages in the study of LROs.