Biochemical Characterization of Molybdenum-Cofactor in a Cyanobacterium, Nostoc muscorum

Cell-free extracts of nitrate-grown Nostoc muscorum containnitrate reductase and molybdenum-cofactor activities. Whilenitrate reductase activity is associated with the paniculatefraction, cofactor activity is found predominantly in the solublefraction. This activity was distributed between two pools. Inone pool, the molybdenumcofactor is associated with a carrier(protein) of approximately 30,000 Da with an S_{20, w} between2.3 and 2.5. The carrier-bound cofactor is non-dialyzable andis found along with the major proteins during filtration inSephadex G-25 and G-100. The second pool contains free or unboundcofactor. It is separated from soluble proteins by dialysiswith a membrane with a pore-size of 10 to 15 kDa. However, itis retained with a membrane with a pore size of 1 kDa. It isin the included volume during chromatography through SephadexG-25. Its molecular mass is estimated to be between 1,000 and5,000 Da. The molybdenum content was proportional to cofactoractivity in both pools. Reducing agents increased cofactor activity.However, activity in both pools was sensitive to heat, acid,and oxidative treatments. The carrier protein appears to givesome protection. ¹Fulbright Scholar from Department of Biological Sciences, R.D. University, Jabalpur-482001, India. To whom reprint requestsshould be addressed. (Received June 22, 1987; Accepted August 21, 1987)     

Activation Energies of Reactions Catalyzed by the Nitrate Reductase from Chlorella vulgaris

The thermal dependence of two of the reactions catalyzed bythe nitrate reductase from Chlorella vulgaris was determined.The activation energies for NADH:nitrate oxidoreductase (EC1.6.6.1 [EC] ) and NADH:Cytochrome c oxidoreductase (EC 1.6.99.3 [EC] )are 42.1 kJ?mol^–1 and 21.5 kJ?mol^–1, respectively.Since the thermal dependency of the two enzymes is different,ratios of the activities will vary with temperature. The importanceof both rigorous thermal control during nitrate reductase assaysas well as the need to specify the temperature at which theratio of activities for the enzyme are clearly established. ¹Present Address: Cropping Systems Research Laboratory, USDA-ARS,Route 3, Box 215, Lubbock, TX 79401, U.S.A. (Received November 25, 1987; Accepted March 2, 1988)     

Phospholipid transfer proteins from lung, properties and possible physiological functions

Phospholipid transfer proteins have been found in lung just as they have in tissues throughout the body. There is speculation that the proteins are involved in membrane biogenesis and in determining the phospholipid composition of membranes. For this reason the lung, which contains subcellular organelles of distinct phospholipid composition, is of interest in terms of its complement of phospholipid transfer proteins. The lamellar bodies of pulmonary type II alveolar cells have a phospholipid composition unique in terms of the proportions of dipalmitoyl phosphatidylcholine and phosphatidylglycerol. Studies of the phospholipid transfer proteins in lung have demonstrated two molecular species of the transfer proteins that differ significantly from those found in liver and other tissues. These proteins show specificity for the transfer of dipalmitoyl phosphatidylcholine and phosphatidylglycerol.     

Fetal lung disaturated phosphatidylcholine. Ostensible increase following exposure to dexamethasone

Fetal rat lung removed at 15 days gestation and placed in organ culture incorporates choline into phosphatidylcholine. Addition of 10(-9) M dexamethasone resulted in increased rates of choline incorporation per micrograms protein after both 6 and 12 days culture. This concentration of dexamethasone did not increase tissue phosphatidylcholine or disaturated phosphatidylcholine. Thus, at a culture time when dexamethasone had a significant effect on choline incorporation, there was no change in either the total phospholipid or disaturated phosphatidylcholine content of the lung tissue. The transplacental administration of dexamethasone decreased fetal lung DNA and phospholipid content. At the mid-range dosage tested (400 micrograms), dexamethasone depressed DNA (51%) appreciably more than total phosphatidylcholine (28%) and disaturated phosphatidylcholine (33%). These results show that the hormone does not increase the total amount of surfactant per lung. The increased disaturated phosphatidylcholine per mg DNA results in an ostensible beneficial effect of dexamethasone on surfactant and may reflect an increased proportion of Type II cells in fetal lung both in vitro and in vivo following hormone exposure. Disaturated phosphatidylcholine per Type II alveolar cell is no doubt increased but the trade-off is fewer total cells in the lung.     

An organ culture system for study of fetal lung development

Fetal rat lungs placed in $\text{in}$$\text{vitro}$ organ culture at 15.5 days gestation grow significantly based on accumulation of DNA and protein. In the experimental system described, DNA accumulated rapidly during the first three days in culture and increased from 4.8 to 15.6 micrograms per lung culture. Protein content increased more slowly and reached a value more than double the initial value after six days in the culture system. Glycogen accumulated in the tissue during the first six days in culture and was depleted during the subsequent culture period, a pattern strikingly similar to that observed during lung development $\text{in}$$\text{vivo}$. Phospholipid accumulation was biphasic with respect to time with an inflection point at about the sixth day of culture. The phosphatidylcholine species synthesized in the culture system $\text{in}$$\text{vitro}$ were similar to those produced $\text{in}$$\text{vivo}$ in fetal lung at 21 days gestation.     

Immunochemical characterization and localization of nitrate reductase in norflurazon-treated soybean cotyledons

Immunochemical procedures were used to characterize and localize NADH:nitrate reductase (NR; EC 1.6.6.1) in cotyledons of norflurazon-treated soybeans [ Glycine max (L.) Merr. cv. 'Hill']. Antiserum prepared to NR isolated from Chlorella strongly reacted against NR from norflurazon-treated cotyledons. This serum inhibited the NR activity in crude extracts of norflurazon-treated soybean cotyledons by 98% even at a 1:2000 dilution of crude serum. Pre-immune serum had no effect on the activity. These data indicate that there are similar antigenic determinants at the active site of both Chlorella and norflurazon-treated soybean NR. Whole cotyledons were homogenized in lithium dodecyl sulfate-containing buffer, electrophoretically separated and blotted to nitrocellulose. When the blots were reacted with the anti-NR serum only a single protein (M_r= 98 kdalton) was visualized. Immunofluorescence studies on fixed tissue sections revealed intense fluorescence in the cytoplasm. Weaker reactions were associated with organelles tentatively identified as plastids. Pre-immune serum controls were completely unstained using immunocytochemical procedures.     

Molecular Subtypes in Head and Neck Cancer Exhibit Distinct Patterns of Chromosomal Gain and Loss of Canonical Cancer Genes

Head and neck squamous cell carcinoma (HNSCC) is a frequently fatal heterogeneous disease. Beyond the role of human papilloma virus (HPV), no validated molecular characterization of the disease has been established. Using an integrated genomic analysis and validation methodology we confirm four molecular classes of HNSCC (basal, mesenchymal, atypical, and classical) consistent with signatures established for squamous carcinoma of the lung, including deregulation of the KEAP1/NFE2L2 oxidative stress pathway, differential utilization of the lineage markers SOX2 and TP63, and preference for the oncogenes PIK3CA and EGFR. For potential clinical use the signatures are complimentary to classification by HPV infection status as well as the putative high risk marker CCND1 copy number gain. A molecular etiology for the subtypes is suggested by statistically significant chromosomal gains and losses and differential cell of origin expression patterns. Model systems representative of each of the four subtypes are also presented.     

Resistance to desiccation in aquatic invasive snails and implications for their overland dispersal

At least 30 species of nonindigenous freshwater snails have invaded North America. The risk of these snails invading new lakes depends upon their ability to survive overland transport. We first reviewed published laboratory experiments using freshwater snails, which show numerous species are able to tolerate days of air exposure. We then tested tolerance to drying of three species of invasive aquatic snails that are widespread in Wisconsin: Bithynia tentaculata, Cipangopaludina chinensis, and Viviparus georgianus. In a series of seven experiments, we simulated boater transport by placing snails individually in mesh bags, hung outdoors, and confined in a screen tent. The screen roof allowed exposure to both sun and rain, and an on-site weather station recorded temperature, precipitation, and humidity. All three species exhibited high survivorship, with some individuals alive at the end of most experiments: 42 days for B. tentaculata and V. georgianus and 63 days for C. chinensis. Viable young were released by C. chinensis after 54 days of exposure. Overall, our results indicate that these invasive snails should readily survive long periods of transport overland, indicating a need for continued vigilance of recreational boaters entering lakes.     

Increased sequence diversity coverage improves detection of HIV-specific T cell responses

The accurate identification of HIV-specific T cell responses is important for determining the relationship between immune response, viral control, and disease progression. HIV-specific immune responses are usually measured using peptide sets based on consensus sequences, which frequently miss responses to regions where test set and infecting virus differ. In this study, we report the design of a peptide test set with significantly increased coverage of HIV sequence diversity by including alternative amino acids at variable positions during the peptide synthesis step. In an IFN-gamma ELISpot assay, these "toggled" peptides detected HIV-specific CD4(+) and CD8(+) T cell responses of significantly higher breadth and magnitude than matched consensus peptides. The observed increases were explained by a closer match of the toggled peptides to the autologous viral sequence. Toggled peptides therefore afford a cost-effective and significantly more complete view of the host immune response to HIV and are directly applicable to other variable pathogens.