Methods of tracing hydrogen in polyketide biosynthesis: High field NMR spectroscopy of citrinin produced in a D2O based medium

Penicillium citrinum cultures have been germinated on an H₂O-based medium, resuspended on a D₂O-based medium and treated with [l,2-¹³C₂] acetate. The resulting citrinin (1) has been analysed by²H and¹³C nuclear magnetic resonance spectroscopy and information about the metabolism of hydrogen in citrinin biosynthesis has been deduced.     

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.     

A molybdopterin-free form of xanthine oxidase

A previously unidentified fraction lacking xanthine:O2 activity has been isolated during affinity chromatography of bovine milk xanthine oxidase preparations on Sepharose 4B/folate gel. Unlike active, desulfo, or demolybdo forms of xanthine oxidase, this form, which typically comprises about 5% of an unfractionated enzyme solution, passes through the affinity column without binding to it, and is thus easily separated from the other species. The absorption spectrum of this fraction is very similar to that of the active form, but has a 7% lower extinction at 450 nm. Analysis of the fraction has shown that it is a dimer of normal size, but that it does not contain molybdenum or molybdopterin (MPT). The "MPT-free" xanthine oxidase contains 90-96% of the Fe found in active xanthine oxidase, and 100% of the expected sulfide. EPR and absorption difference spectroscopy indicate that the MPT-free fraction is missing approximately half of its Fe/S I centers. The presence of a new EPR signal suggests that an altered Fe/S center may account for the nearly normal Fe and sulfide content. Microwave power saturation parameters for the Fe/S II and Fe/S I centers in the MPT-free fraction are normal, with P1/2 equal to 1000 and 60 mW, respectively. The new EPR signal shows intermediate saturation behavior with a P1/2 = 200 mW. The circular dichroism spectrum of the MPT-free fraction shows distinct differences from that of active enzyme. The NADH:methylene blue activity of the MPT-free fraction is the same as that of active xanthine oxidase which exhibits xanthine:O2 activity, but NADH:cytochrome c and NADH:DCIP activities are diminished by 54 and 37%, respectively.     

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)