Radiation inactivation analysis of assimilatory NADH:nitrate reductase. Apparent functional sizes of partial activities associated with intact and proteolytically modified enzyme

Recently we demonstrated that target sizes for the partial activities of nitrate reductase were considerably smaller than the 100-kDa subunit which corresponded to the target size of the full (physiologic) activity NADH:nitrate reductase. These results suggested that the partial activities resided on functionally independent domains and that radiation inactivation may be due to localized rather than extensive damage to protein structure. The present study extends these observations and addresses several associated questions. Monophasic plots were observed over a wide range of radiation doses, suggesting a single activity component in each case. No apparent differences were observed over a 10-fold range of concentration for each substrate, suggesting that the observed slopes were not due to marked changes in Km values. Apparent target sizes estimated for partial activities associated with native enzyme and with limited proteolysis products of native enzyme suggested that the functional size obtained by radiation inactivation analysis is independent of the size of the polypeptide chain. The presence of free radical scavengers during irradiation reduced the apparent target size of both the physiologic and partial activities by an amount ranging from 24 to 43%, suggesting that a free radical mechanism is at least partially responsible for the inactivation. Immunoblot analysis of nitrate reductase irradiated in the presence of free radical scavengers revealed formation of distinct bands at 90, 75, and 40 kDa with increasing doses of irradiation rather than complete destruction of the polypeptide chain.     

Characterization of the chicken oocyte receptor for low and very low density lipoproteins

The chicken oocyte receptor for low and very low density lipoproteins has been identified and characterized. Receptor activity present in octyl-beta-D-glucoside extracts of oocyte membranes was measured by a solid phase filtration assay, and the receptor was visualized by ligand blotting. The protein had an apparent Mr of 95,000 in sodium dodecyl sulfate-polyacrylamide gels under nonreducing conditions and exhibited high affinity for apolipoprotein B-containing lipoproteins, but not for high density lipoproteins or lipoproteins in which lysine residues had been reductively methylated. Binding of lipoproteins was sensitive to EDTA, suramin, and treatment with Pronase. In these aspects, the avian oocyte system was analogous to the mammalian low density lipoprotein receptor in somatic cells. Furthermore, a structural relationship between the mammalian and avian receptors was revealed by immunoblotting: polyclonal antibodies directed against the purified bovine low density lipoprotein receptor reacted selectively with the 95-kDa chicken receptor present in crude oocyte membrane extracts.     

Effect of a hyperlipidic diet on lipid composition, fluidity, and (Na+-K+)ATPase activity of rat erythrocyte membranes

Feeding rats a hyperlipidic diet in which animals were offered daily a variety of high-energy food resulted in a significant increase of serum free fatty acids and a decrease of phospholipids with respect to controls. On the contrary, there were no significant differences in erythrocyte membrane total lipid composition between the two groups. Erythrocyte membranes showed a significant decrease in saturated fatty acid content and a significant increase in (n-6) polyunsaturated fatty acid content; (n-3) polyunsaturated fatty acids significantly decreased. Membrane fluidity, investigated by fluorescence polarization of diphenylhexatriene, significantly increased in the erythrocyte membranes of the experimental group. These results seem compatible with the decreased saturated/unsaturated fatty acid ratio. A significant decrease of (Na+-K+)ATPase activity occurred in erythrocyte membranes of the experimental group rats with respect to the controls.     

Haemogregarina georgianae n. sp., a new haemogregarine parasite from the Antarctic teleost Parachaenichthys georgianus

Haemogregarina georgianae n. sp. occurs frequently in the bathydraconid teleost Parachaenichthys georgianus from the western Antarctic portion of the Southern Ocean. The haemogregarine and its developmental stages in the vertebrate have been found in erythrocytes of the fish: both microschizogony and macroschizogony have been seen in fish caught during the austral summer. Morphological evidence suggests the merozoites from macroschizogony give rise to the microschizont, and that the microschizont merozoites give rise to gametocytes. Comparison of H. georgianae with other haemogregarines known from teleosts shows that it has a previously undescribed morphology.     

Seroreactivity to HPV-16 proteins in women with early cervical neoplasia

Summary Although serological reactivity to human papillomavirus type 16 (HPV-16) proteins has been demonstrated in patients with invasive cervical carcinoma, the degree of seroreactivity to these proteins in women with preinvasive disease and its relationship to the HPV type associated with the disease are unclear. We obtained sera from 27 women undergoing cone biopsy for cervical precursor lesions and 22 controls and analyzed seroreactivity by Western blot to fusion proteins containing portions of the HPV-16 E4, L1 and L2 open-reading frames (ORFs). Positives were analyzed by scanning densitometry and intensity values for each case plotted relative to controls. Cervical biopsy specimens from patients were analyzed for HPV-16 nucleic acids by DNA · DNA in situ hybridization. Mean intensity values for seroreactivity to the pATH-E4 protein approached significance (P = 0.058) and a significantly higher proportion of cases vs controls registered values over 4.0 for pATH-E4 (26% vs 4.5%;P = 0.04) and pATH-L2 (48% vs 18%;P = 0.03) proteins. A significantly higher mean intensity value for E4 was observed for cases containing HPV-16 DNA vs HPV-16 negative cases or controls. Thus, seroreactivity to HPV-16-derived proteins may be more common in women with preinvasive cervical disease, and for some protein targets (E4) may indicate a relatively type-specific response.Supported in part by grants from the National Cancer Institute [CA 47676 (C.P.C.)], American Cancer Society [MV-395 (C.P.C.)] and an institutional support grant (J.K.R.). Dr. Crum is a recipient of a Physician Scientist Award from the National Institute of Allergy and Infectious Disease (AI00628)     

Vitamin contents of archaebacteria.

The levels of six water-soluble vitamins of seven archaebacterial species were determined and compared with the levels found in a eubacterium, Escherichia coli. Biotin, riboflavin, pantothenic acid, nicotinic acid, pyridoxine, and lipoic acid contents of Halobacterium volcanii, Methanobacterium thermoautotrophicum delta H, "Archaeoglobus fulgidus" VC-16, Thermococcus celer, Pyrodictium occultum, Thermoproteus tenax, and Sulfolobus solfataricus were measured by using bioassays. The archaebacteria examined were found to contain these vitamins at levels similar to or significantly below the levels found in in E. coli. Riboflavin was found at levels comparable to those in E. coli. Pyridoxine was as abundant among the archaebacteria of the methanogenhalophile branch as in E. coli. It was only one-half as abundant in the sulfur-metabolizing branch. "A. fulgidus," however, contained only 4% as much pyridoxine as E. coli. Nicotinic and pantothenic acids were approximately 10-fold less abundant (except for a 200-fold-lower nicotinic acid level in "A. fulgidus"). Nicotinic acid may be replaced by an 8-hydroxy-5-deazaflavin coenzyme (factor F420) in some archaebacteria (such as "A. fulgidus"). Compared with the level in E. coli, biotin was equally as abundant in Thermococcus celer and Methanobacterium thermoautotrophicum, about one-fourth less abundant in P. occultum and "A. fulgidus," and 25 to over 100 times less abundant in the others. The level of lipoic acid was up to 20 times lower in H. volcanii, Methanobacterium thermoautotrophicum, and Thermococcus celer. It was over two orders of magnitude lower among the remaining organisms. With the exception of "A. fulgidus," lipoic acid, pantothenic acid, and pyridoxine were more abundant in the members of the methanogen-halophile branch of the archaebacteria than in the sulfur-metabolizing branch.     

Genetic analysis of blood pressure in the Milan hypertensive strain of rat (Rattus norvegicus)

Estimates of heritability (h2) of blood pressure level and the number of loci controlling the trait were derived from two genetic crosses involving the Milan hypertensive strain of rat and its control with normal blood pressure. In the genetic cross involving backcrosses, the estimates were h2 = 64% and the number of loci was two or three; there was some evidence of dominance of the alleles for normal blood pressures. In the other cross with only F2's, the degree of genetic determination (heritability in the broad sense) was 45%, involving at least three loci.     

Effect of soil moisture and phosphate level on root hair growth of corn roots

Summary Root hairs have been shown to enhance P uptake by plants growing in low P soil. Little is known of the factors controlling root hair growth. The objective of this study was to investigate the influence of soil moisture and P level on root hair growth of corn (Zea mays L.). The effect of volumetric soil moistures of 22% (M₀), 27% (M₁), and 32% (M₂) and soil (Raub silt loam, Aquic Argiudoll) P levels of, 0.81 (P₀), 12.1 (P₁), 21.6 (P₂), 48.7 (P₃), and 203.3 (P₄) mol P L^–1 initially in the soil solution, on shoot and root growth, P uptake, and root hair growth of corn was studied in a series of pot experiments in a controlled climate chamber. Root hair growth was affected more by soil moisture than soil P. The percentage of total root length with root hairs and the density and length of root hairs on the root sections having root hairs all increased as soil moisture was reduced from M₂ to M₀. No relationship was found between root hair length and soil P. Density of root hairs, however, was found to decrease with an increase in soil P. No correlation was found between root hair growth parameters and plant P content, further suggesting P plays a secondary role to moisture in regulating root hair growth in soils. The increase in root hair growth appears to be a response by the plant to stress as yield and P uptake by corn grown at M₀ were only 0.47 to 0.82, and 0.34 to 0.74, respectively, of that measured at M₁ across the five soil P levels. The increase in root hair growth at M₀, which represents an increase of 2.76 to 4.03 in root surface area, could offset, in part, the reduced rate of root growth, which was the primary reason for reduced P uptake under limited soil moisture conditions.Journal Paper No. 10,066 Purdue Univ. Agric. Exp. Stn., W. Lafayette, IN 47907. Contribution from the Dep. of Agron. This paper was supported in part by a grant from the Tennessee Valley Authority.