Purification and characterization of RNA polymerase I from a higher plant.

RNA polymerase I was purified from chromatin isolated from auxin-treated soybean hypocotyl. Purification was achieved by using Agarose A-1.5m gel filtration, DEAE-cellulose, CM-sephadex, and phosphocellulose chromatography, and sucrose density gradient centrifugation. With denatured calf thymus DNA as template, the enzyme has a high specific activity (200-300 nmol/mg/30 min at 28 degrees C) which is comparable to other RNA polymerase I enzymes purified from animals and yeast. While the gel profiles indicate that purification to homogeneity (greater than 90%) may not have been achieved, the enzyme appears to be composed of possibly 7 subunits, several of which are similar to the subunits of yeast RNA polymerase I. The putative subunits and molar ratios are 183 000 (1), 136 000 (1), 50 000 (0.5), 46 000 (0.5), 40 000 (0.5), 33 000 (0.2), and 28 000 (2). The purified enzyme strongly prefers a completely denatured template such as poly(dC).     

Novel Roles for Kv7 Channels in Shaping Histamine-Induced Contractions and Bradykinin-Dependent Relaxations in Pig Coronary Arteries

Voltage-gated K_v7 channels are inhibited by agonists of G_q-protein-coupled receptors, such as histamine. Recent works have provided evidence that inhibition of vascular K_v7 channels may trigger vessel contractions. In this study, we investigated how K_v7 activity modulates the histamine-induced contractions in “healthy” and metabolic syndrome (MetS) pig right coronary arteries (CAs). We performed isometric tension and immunohistochemical studies with domestic, lean Ossabaw, and MetS Ossabaw pig CAs. We found that neither the K_v7.2/K_v7.4/K_v7.5 activator ML213 nor the general K_v7 inhibitor XE991 altered the tension of CA rings under preload, indicating that vascular K_v7 channels are likely inactive in the preloaded rings. Conversely, ML213 potently dilated histamine-pre-contracted CAs, suggesting that K_v7 channels are activated during histamine applications and yet partially inhibited by histamine. Immunohistochemistry analysis revealed strong K_v7.4 immunostaining in the medial and intimal layers of the CA wall, whereas K_v7.5 immunostaining intensity was strong in the intimal but weak in the medial layers. The medial K_v7 immunostaining was significantly weaker in MetS Ossabaw CAs as compared to lean Ossabaw or domestic CAs. Consistently, histamine-pre-contracted MetS Ossabaw CAs exhibited attenuated ML213-dependent dilations. In domestic pig CAs, where medial K_v7 immunostaining intensity was stronger, histamine-induced contractions spontaneously decayed to ~31% of the peak amplitude within 4 minutes. Oppositely, in Ossabaw CAs, where K_v7 immunostaining intensity was weaker, the histamine-induced contractions were more sustained. XE991 pretreatment significantly slowed the decay rate of histamine-induced contractions in domestic CAs, supporting the hypothesis that increased K_v7 activity correlates with a faster rate of histamine-induced contraction decay. Alternatively, XE991 significantly decreased the amplitude of bradykinin-dependent dilations in pre-contracted CAs. We propose that in CAs, a decreased expression or a loss of function of K_v7 channels may lead to sustained histamine-induced contractions and reduced endothelium-dependent relaxation, both risk factors for coronary spasm.     

Characterization of cDNA of lycopene beta-cyclase responsible for a high level of beta-carotene accumulation in Dunaliella salina.

Lycopene beta-cyclase (Lyc-B) is the key enzyme in the catalysis of linear lycopene to form cyclic beta-carotene, an indispensable part of the photosynthetic apparatus and an important source of vitamin A in human and animal nutrition. Studies showing that the microalga Dunaliella salina can accumulate a high level of beta-carotene are lacking. We hypothesize that D. salina is closely involved with the catalytic mechanism of Lyc-B and the molecular regulation of its gene. In this study, we used RT-PCR and RACE-PCR to isolate a 2475 bp cDNA with a 1824 bp open reading frame, encoding a putative Lyc-B, from D. salina. Homology studies showed that the deduced amino acid sequence had a significant overall similarity with sequences of other green algae and higher plants, and that it shared the highest sequence identity, up to 64%, with Lyc-B of Chlamydomonas reinhardtii. Codon analysis showed that synonymous codon usage in the enzyme has a strong bias towards codons ending with adenosine. Two motifs were found in the Lyc-B sequence, one at the N terminus, for binding the hypothetical cofactor FAD, and the other was a substrate carrier motif in oxygenic organisms shared by an earlier carotenogenesis enzyme, phytoene desaturase, and Lyc-B. A tertiary structure prediction suggested that the catalytic or binding site structure within LycB from D. salina is superior to that of both H. pluvialis and C. reinhardtii. The LycB protein from D. salina was quite removed from that of H. pluvialis and C. reinhardtii in the phylogenetic tree. Taken as a whole, this information provides insight into the regulatatory mechanism of Lyc-B at the molecular level and the high level of beta-carotene accumulation in the microalga D. salina.     

Simultaneous degradation of organophosphorus pesticides and p-nitrophenol by a genetically engineered Moraxella sp. with surface-expressed organophosphorus hydrolase.

Moraxella sp., a native soil organism that grows on p-nitrophenol (PNP), was genetically engineered for the simultaneous degradation of organophosphorus (OP) pesticides and p-nitrophenol (PNP). The truncated ice nucleation protein (INPNC) anchor was used to target the pesticide-hydrolyzing enzyme, organophosphorus hydrolase (OPH), onto the surface of Moraxella sp., alleviating the potential substrate uptake limitation. A shuttle vector, pPNCO33, coding for INPNC-OPH was constructed and the translocation, surface display, and functionality of OPH were demonstrated in both E. coli and Moraxella sp. However, whole cell activity was 70-fold higher in Moraxella sp. than E. coli. The resulting Moraxella sp. degraded organophosphates as well as PNP rapidly, all within 10 h. The initial hydrolysis rate was 0.6 micromol/h/mg dry weight, 1.5 micromol/h/mg dry weight, and 9.0 micromol/h/mg dry weight for methyl parathion, parathion, and paraoxon, respectively. The possibility of rapidly degrading OP pesticides and their byproducts should open up new opportunities for improved remediation of OP nerve agents in the future.     

BJ-HCC-20, a potential novel cancer-testis antigen.

In an effort to identify novel Cancer-Testis genes, we analyzed the sequence in the q26-28 region of human X chromosome by several on-line tools. The candidate sequences were then confirmed by experiments. We have obtained a novel Cancer-Testis gene, BJ-HCC-20. In vivo, it was found to have two isoforms. In samples of liver, colon, gastric and lung cancer tested, the expression frequency of BJ-HCC-20 is 25%, 17%, 21% and 15%, respectively. Full-length cDNAs of both BJ-HCC-20 isoforms were isolated and their gene structures and promoter regions were characterized. BJ-HCC-20 might have implications in theoretical and practical tumor biology.     

Fibronectin-degrading proteases from the membranes of transformed cells

The local degradation of fibronectin substrata by Rous sarcoma virus-transformed chick embryonic fibroblasts requires cell-contact-related metalloendoprotease and serine-protease activities. Using fibronectin-containing SDS gels, two large proteases with apparent molecular weights of 120K and 150K were found only in the membrane fraction of transformed cells and were absent in normal cells. Both 120K and 150K proteases were active at neutral pH, but showed preferential inhibitor sensitivities of serine and metal proteases, respectively. The 150K protease appeared to account for most of the proteolytic activity since metalloendoprotease inhibitors completely blocked proteolytic activity of the 150K in fibronectin gels, more than 80% of the fibronectin-degrading activity of solubilized membranes, and largely suppressed the appearance of fibronectin degradation spots in cultures of transformed cells.     

Phosphodiester bond cleavage outside mitochondria is required for the completion of protein import into the mitochondrial matrix

The present studies show that hydrolysis of a phosphodiester bond, most likely ATP, is a distinct, second step required to complete import of the F1-ATPase beta-subunit into the mitochondria. This step follows a membrane potential-dependent first step. We show, using an inhibitor of adenine nucleotide transport and the analogue beta,gamma-AMP-PCP, that the activity required for this phosphodiester hydrolysis-dependent completion of protein import resides outside the mitochondrial inner membrane. This activity is proposed to act on the precursor at the site of translocation either to render it competent or to catalyze its vectorial movement directly through the import apparatus. This activity shares properties ascribed to proteins of the heat-shock family, which are proposed to participate in the ATP-dependent refolding of partially denatured proteins and nascent peptides.     

Identification of HLA-B44 subtypes associated with extended MHC haplotypes

The HLA class I antigen B44 is found in each of two different extended major histocompatibility haplotypes (allele combinations of HLA-B, HLA-DR, and complement genes BF, C2, C4A, and C4B in linkage disequilibrium). Using isoelectric focusing, two variants of HLA-B44 were identified. The basic variant was found in all cell lines with the extended haplotype HLA-B44, DR7, FC31, and the acidic variant in all cell lines with the extended haplotype HLA-B44, DR4, SC30. The occurrence of each antigen variant with a unique extended haplotype explains previous observations concerning the nonrandom association of B44 variants with DR antigens.     

Effect of hydrophobicity of utilization of peptides by ruminal bacteria in vitro

When mixed ruminal bacteria were incubated with a pancreatic casein hydrolysate and free amino acids of a similar composition, rates of ammonia production were much greater for peptides than for amino acids. The pancreatic digest of casein was then fractionated with 90% isopropyl alcohol. Hydrophobic peptides which dissolved in alcohol contained an abundance of phenolic and aliphatic amino acids, while the hydrophilic peptides which were precipitated by alcohol contained a large proportion of the highly charged amino acids. The Km values of the mixed ruminal bacteria for each fraction were similar (0.88 versus 0.98 g/liter), but the Vmax of the hydrophilic peptides was more than twice that of the hydrophobic peptides (18 versus 39 mg of NH3 per g of bacterial protein per h). Pure cultures of ruminal bacteria had a similar preference for hydrophilic peptides and likewise utilized peptides at a faster rate than free amino acids. Since peptide degradation rates differed greatly, hydrophobicity is likely to influence the composition of amino acids passing unfermented to the lower gut of ruminant animals.