Developmental regulation of the gene for formate dehydrogenase in Neurospora crassa.

We have isolated and characterized a gene, fdh, from Neurospora crassa which is developmentally regulated and which produces formate dehydrogenase activity when expressed in Escherichia coli. The gene is closely linked (less than 0.6 kb apart) to the leu-5 gene encoding mitochondrial leucyl-tRNA synthetase; the two genes are transcribed convergently from opposite strands. The expression patterns of these genes differ: fdh mRNA is found only during conidiation and early germination and is not detectable during mycelial growth, while leu-5 mRNA appears during germination and mycelial growth. The structure of the fdh gene was determined from the sequence of cDNA and genomic DNA clones and from mRNA mapping studies. The gene encodes a 375-amino-acid-long protein with sequence similarity to NAD-dependent dehydrogenases of the E. coli 3-phosphoglycerate dehydrogenase (serA gene product) subfamily. In particular, there is striking sequence similarity (52% identity) to formate dehydrogenase from Pseudomonas sp. strain 101. All of the residues thought to interact with NAD in the crystal structure of the Pseudomonas enzyme are conserved in the N. crassa enzyme. We have further shown that expression of the N. crassa gene in E. coli leads to the production of formate dehydrogenase activity, indicating that the N. crassa gene specifies a functional polypeptide.     

Experimental and Computational Investigation of Biofilm Formation by Rhodopseudomonas palustris Growth under Two Metabolic Modes

We examined biofilms formed by the metabolically versatile bacterium Rhodopseudomonas palustris grown via different metabolic modes. R. palustris was grown in flow cell chambers with identical medium conditions either in the presence or absence of light and oxygen. In the absence of oxygen and the presence of light, R. palustris grew and formed biofilms photoheterotrophically, and in the presence of oxygen and the absence of light, R. palustris grew and formed biofilms heterotrophically. We used confocal laser scanning microscopy and image analysis software to quantitatively analyze and compare R. palustris biofilm formation over time in these two metabolic modes. We describe quantifiable differences in structure between the biofilms formed by the bacterium grown heterotrophically and those grown photoheterotrophically. We developed a computational model to explore ways in which biotic and abiotic parameters could drive the observed biofilm architectures, as well as a random-forest machine-learning algorithm based on structural differences that was able to identify growth conditions from the confocal imaging of the biofilms with 87% accuracy. Insight into the structure of phototrophic biofilms and conditions that influence biofilm formation is relevant for understanding the generation of biofilm structures with different properties, and for optimizing applications with phototrophic bacteria growing in the biofilm state.     

The effect of different spectral light quality on the photoinhibition of Photosystem I in intact leaves

Photosynthesis Research - Light energy causes damage to Photosystem I (PSI) and Photosystem II (PSII). The majority of the previous photoinhibition studies have been conducted with PSII, which...     

Influence of Formulation and Processing Variables on Properties of Itraconazole Nanoparticles Made by Advanced Evaporative Precipitation into Aqueous Solution

Nanoparticles, of the poorly water-soluble drug, itraconazole (ITZ), were produced by the Advanced Evaporative Precipitation into Aqueous Solution process (Advanced EPAS). This process combines emulsion templating and EPAS processing to provide improved control over the size distribution of precipitated particles. Specifically, oil-in-water emulsions containing the drug and suitable stabilizers are sprayed into a heated aqueous solution to induce precipitation of the drug in form of nanoparticles. The influence of processing parameters (temperature and volume of the heated aqueous solution; type of nozzle) and formulation aspects (stabilizer concentrations; total solid concentrations) on the size of suspended ITZ particles, as determined by laser diffraction, was investigated. Furthermore, freeze-dried ITZ nanoparticles were evaluated regarding their morphology, crystallinity, redispersibility, and dissolution behavior. Results indicate that a robust precipitation process was developed such that size distribution of dispersed nanoparticles was shown to be largely independent across the different processing and formulation parameters. Freeze-drying of colloidal dispersions resulted in micron-sized agglomerates composed of spherical, sub-300-nm particles characterized by reduced crystallinity and high ITZ potencies of up to 94% (w/w). The use of sucrose prevented particle agglomeration and resulted in powders that were readily reconstituted and reached high and sustained supersaturation levels upon dissolution in aqueous media.     

Mutations that specifically impair the DNA binding activity of the herpes simplex virus protein UL42.

The herpes simplex virus DNA polymerase is a heterodimer consisting of a catalytic subunit and the protein UL42, which functions as a processivity factor. It has been hypothesized that UL42 tethers the catalytic subunit to the DNA template by virtue of DNA binding activity (J. Gottlieb, A. I. Marcy, D. M. Coen, and M. D. Challberg, J. Virol. 64:5976-5987, 1990). Relevant to this hypothesis, we identified two linker insertion mutants of UL42 that were unable to bind to a double-stranded-DNA-cellulose column but retained their ability to bind the catalytic subunit. These mutants were severely impaired in the stimulation of long-chain-DNA synthesis by the catalytic subunit in vitro. In transfected cells, the expressed mutant proteins localized to the nucleus but were nonetheless deficient in complementing the growth of a UL42 null virus. Thus, unlike many other processivity factors, UL42 appears to require an intrinsic DNA binding activity for its function both in vitro and in infected cells. Possible mechanisms for the activity of UL42 and its potential as a drug target are discussed.     

Seroepidemiology of Coxsackievirus A6, Coxsackievirus A16, and Enterovirus 71 Infections among Children and Adolescents in Singapore, 2008-2010

Coxsackieviruses A6 (CV-A6) and A16 (CV-A16) and Enterovirus 71 (EV-A71) have caused periodic epidemics of hand, foot and mouth disease (HFMD) among children in Singapore. We conducted a cross-sectional study to estimate the seroprevalence of these enteroviruses among Singapore children and adolescents. The study was conducted between August 2008 and July 2010. It involved 700 Singapore residents aged 1–17 years whose residual sera were obtained following the completion of routine biochemical investigations in two public acute-care hospitals. The levels of neutralizing antibodies (NtAb) against CV-A6, CV-A16 and EV-A71 were analyzed by the microneutralization test. The age-specific geometric mean titer (GMT) of antibodies against each of the three enteroviruses and the 95% confidence intervals (CI) were calculated. The seroprevalence of CV-A6 and CV-A16 was high at 62.7% (95% CI: 59.1–66.2%) and 60.6% (95% CI: 56.9–64.1%), respectively. However, the seroprevalence of EV-A71 was significantly lower at 29.3% (95% CI: 26.0–32.8%). About 89.7% of the children and adolescents had been infected by at least one of the three enteroviruses by 13–17 years of age. About half (52.3%) were seropositive for two or all three enteroviruses, while only 16.1% had no NtAb against any of the three enteroviruses. High NtAb levels were observed in the younger age groups. CV-A6 and CV-A16 infections are very common among Singapore children and adolescents, while EV-A71 infections are less common. Infection is continually acquired from early childhood to adolescent age.     

Human immunodeficiency virus type 1 Vpr interacts with HHR23A, a cellular protein implicated in nucleotide excision DNA repair.

The human immunodeficiency virus type 1 (HIV-1) vpr gene is an evolutionarily conserved gene among the primate lentiviruses HIV-1, HIV-2, and simian immunodeficiency viruses. One of the unique functions attributed to the vpr gene product is the arrest of cells in the G2 phase of the cell cycle. Here we demonstrate that Vpr interacts physically with HHR23A, one member of an evolutionarily conserved gene family involved in nucleotide excision repair. Interaction of Vpr with HHR23A was initially identified through a yeast two-hybrid screen and was confirmed by the demonstration of direct binding between bacterially expressed recombinant and transiently expressed or chemically synthesized protein products. Visualization of HHR23A and Vpr by indirect immunofluorescence and confocal microscopy indicates that the two proteins colocalize at or about the nuclear membrane. We also map the Vpr-binding domain in HHR23A to a C-terminal 45-amino-acid region of the protein previously shown to have homology to members of the ubiquitination pathway. Overexpression of HHR23A and a truncated derivative which includes the Vpr-binding domain results in a partial alleviation of the G2 arrest induced by Vpr, suggesting that the interaction between Vpr and HHR23A is critical for cell cycle arrest induced by Vpr. These results provide further support for the hypothesis that Vpr interferes with the normal function of a protein or proteins involved in the DNA repair process and, thus, in the transmission of signals that allow cells to transit from the G2 to the M phase of the cell cycle.     

Characterization of a novel alpha1,2-fucosyltransferase of Escherichia coli O128:b12 and functional investigation of its common motif

The wbsJ gene from Escherichia coli O128:B12 encodes an alpha1,2-fucosyltransferase responsible for adding a fucose onto the galactose residue of the O-antigen repeating unit via an alpha1,2 linkage. The wbsJ gene was overexpressed in E. coli BL21 (DE3) as a fusion protein with glutathione S-transferase (GST) at its N-terminus. GST-WbsJ fusion protein was purified to homogeneity via GST affinity chromatography followed by size exclusion chromatography. The enzyme showed broad acceptor specificity with Galbeta1,3GalNAc (T antigen), Galbeta1,4Man and Galbeta1,4Glc (lactose) being better acceptors than Galbeta-O-Me and galactose. Galbeta1,4Fru (lactulose), a natural sugar, was furthermore found to be the best acceptor for GST-WbsJ with a reaction rate four times faster than that of lactose. Kinetic studies showed that GST-WbsJ has a higher affinity for lactose than lactulose with apparent Km values of 7.81 mM and 13.26 mM, respectively. However, the kcat/appKm value of lactose (6.36 M(-1) x min(-1)) is two times lower than that of lactulose (13.39 M(-1) x min(-1)). In addition, the alpha1,2-fucosyltransferase activity of GST-WbsJ was found to be independent of divalent metal ions such as Mn2+ or Mg2+. This activity was competitively inhibited by GDP with a Ki value of 1.41 mM. Site-directed mutagenesis and a GDP-bead binding assay were also performed to investigate the functions of the highly conserved motif H152xR154R155xD157. In contrast to alpha1,6-fucosyltransferases, none of the mutants of WbsJ within this motif exhibited a complete loss of enzyme activity. However, residues R154 and D157 were found to play critical roles in donor binding and enzyme activity. The results suggest that the common motif shared by both alpha1,2-fucosyltransferases and alpha1,6-fucosyltransferases have similar functions. Enzymatic synthesis of fucosylated sugars in milligram scale was successfully performed using Galbeta-O-Me and Galbeta1,4Glcbeta-N3 as acceptors.     

Efficiency of peptide nucleic acid-directed PCR clamping and its application in the investigation of natural diets of the Japanese eel leptocephali

Polymerase chain reaction (PCR)-clamping using blocking primer and DNA-analogs, such as peptide nucleotide acid (PNA), may be used to selectively amplify target DNA for molecular diet analysis. We investigated PCR-clamping efficiency by studying PNA position and mismatch with complementary DNA by designing PNAs at five different positions on the nuclear rDNA internal transcribed spacer 1 of the Japanese eel Anguilla japonica in association with intra-specific nucleotide substitutions. All five PNAs were observed to efficiently inhibit amplification of a fully complementary DNA template. One mismatch between PNA and template DNA inhibited amplification of the template DNA, while two or more mismatches did not. DNA samples extracted from dorsal muscle and intestine of eight wild-caught leptochephalus larvae were subjected to this analysis, followed by cloning, nucleotide sequence analysis, and database homology search. Among 12 sequence types obtained from the intestine sample, six were identified as fungi. No sequence similarities were found in the database for the remaining six types, which were not related to one another. These results, in conjunction with our laboratory observations on larval feeding, suggest that eel leptocephali may not be dependent upon living plankton for their food source.