Isolation of subfractions of human very low density lipoproteins by zonal ultracentrifugation.

Very low density lipoproteins (VLDL) have been isolated and subfractionated on the basis of their differing flotation rates. The procedure consists of a single 45-min zonal ultracentrifugation step using a linear density gradient of d = 1.00 to 1.15 g/ml. Appropriate fractions of the zonal rotor effluent containing the entire VLDL spectrum were characterized by analytical ultracentrifugation, gel filtration chromatography, and complete chemical analysis. Flotation rates of VLDL subspecies from hypertriglyceridemic and normolipemic plasmas correlated directly with their Stokes radii and triglyceride content and inversely with their proportion of cholesterol, cholesteryl esters, phospholipids, and total protein. There was also an inverse correlation of flotation rate with the fraction of tetramethylurea-insoluble protein. This procedure provides a reliable methodology for a rapid isolation of VLDL subfractions and the accurate determination of their flotation rates.     

Characterization of leucocin A-UAL 187 and cloning of the bacteriocin gene from Leuconostoc gelidum.

Leucocin A-UAL 187 is a bacteriocin produced by Leuconostoc gelidum UAL 187, a lactic acid bacterium isolated from vacuum-packaged meat. The bacteriocin was purified by ammonium sulfate or acid (pH 2.5) precipitation, hydrophobic interaction chromatography, gel filtration, and reversed-phase high-performance liquid chromatography with a yield of 58% of the original activity. Leucocin A is stable at low pH and heat resistant, and the activity of the pure form is enhanced by the addition of bovine serum albumin. It is inactivated by a range of proteolytic enzymes. The molecular weight was determined by mass spectrometry to be 3,930.3 +/- 0.4. Leucocin A-UAL 187 contains 37 amino acids with a calculated molecular weight of 3,932.3. A mixed oligonucleotide (24-mer) homologous to the sequence of the already known N terminus of the bacteriocin hybridized to a 2.9-kb HpaII fragment of a 7.6-MDa plasmid from the producer strain. The fragment was cloned into pUC118 and then subcloned into a lactococcal shuttle vector, pNZ19. DNA sequencing revealed an operon consisting of a putative upstream promoter, a downstream terminator, and two open reading frames flanked by a putative upstream promoter and a downstream terminator. The first open reading frame downstream of the promoter contains 61 amino acids and is identified as the leucocin structural gene, consisting of a 37-amino-acid bacteriocin and a 24-residue N-terminal extension. No phenotypic expression of the bacteriocin was evident in several lactic acid bacteria that were electrotransformed with pNZ19 containing the 2.9-kb cloned fragment of the leucocin A plasmid.     

Diethyldithiocarbamate Potentiates the Neurotoxicity of In Vivo l-Methyl-4-Phenyl-1, 2, 3, 6-Tetrahydropyridine and of In Vitro 1-Methyl-4-Phenylpyridinium

Diethyldithiocarbamic acid (DDC) potentiates in vivo neurotoxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and in vitro neurotoxicity of 1-methyl-4-phenylpyridinium (MPP+). Male C57B1/6 mice were given two or five injections of MPTP (30 mg/kg i.p.) preceded 0.5 h by DDC (400 mg/kg i.p.). The mice were tested for catalepsy, akinesia, or motor activity during and after the period of dosing. Striatal and hippocampal tissues were obtained at 2 and 7 days following the last injection and evaluated for dopamine and norepinephrine levels, respectively. These same tissues were also analyzed for the levels of glial fibrillary acidic protein (GFAP), an astrocyte-localized protein known to increase in response to neural injury. Pretreatment with DDC potentiated the effect of MPTP in striatum and resulted in substantially greater dopamine depletion, as well as a more pronounced elevation in GFAP. In hippocampus, the levels of norepinephrine and GFAP were not different from controls in mice receiving only MPTP, but pretreatment with DDC resulted in a sustained depletion of norepinephrine and an elevation of GFAP, suggesting that damage was extended to this brain area by the combined treatment. Mice receiving MPTP preceded by DDC also demonstrated a more profound, but reversible, catalepsy and akinesia compared to those receiving MPTP alone. Systemically administered MPP+ decreased heart norepinephrine, but did not alter the striatal levels of dopamine or GFAP, and pretreatment with DDC did not alter these effects, but did increase lethality. DDC is known to increase brain levels of MPP+ after MPTP, but our data indicate that this is not due to a movement of peripherally generated MPP+ into CNS.(ABSTRACT TRUNCATED AT 250 WORDS)     

Archaeoparasitology in North America

The study of prehistoric parasitism through analysis of coprolites, mummies, skeletons, and latrine soils is rapidly growing. Its development in North America is interdisciplinary and is derived from the fields of physical anthropology, parasitology, and archaeology. The various parasite finds from North America are reviewed. The data show that prehistoric peoples in North America suffered from a variety of parasitic diseases. The validity of the findings are then considered. Although most finds of parasites from prehistoric contexts result from human infections, some finds cannot be verified as such. However, in combination with data from South America, it is clear that prehistoric peoples in the Americas were host to a variety of human parasites, some of which were not previously thought to be present before historic times.     

Conjugal Transfer of Megaplasmid 2 between Rhizobium meliloti Strains in Alfalfa Nodules

A DNA fragment containing the RP4 mob function, as well as the gentamicin and spectinomycin resistance genes, was inserted by gene replacement onto the megaplasmid 2 (pM2) of Rhizobium meliloti 0540 (Inf⁻ EPS⁻), resulting in PG101 (Inf⁻ EPS⁻). The self-transfer of pM2 and the mobilization of pM2 by plasmid RP4-4 were investigated during conjugation between PG101 and R. meliloti 2526 (Nod⁻). In filter conjugations, pM2 was readily mobilized by RP4-4. In addition to this, the self-transfer of one megaplasmid (pM) was detected at a frequency of 3 × 10⁻⁷. Bacteria isolated from the nodules of alfalfa and coinoculated with strains PG101 and 2526 showed that pM2 was mobilized at a frequency of approximately 7 × 10⁻⁵. Bacterial cell numbers were too low in the nodules for detection of the self-transfer of pM2 to occur. No pM2 transfer was detected in the inoculum. A comparison of the transfer frequencies for the various conjugation conditions revealed that pM2 transfer occurred as frequently in the nodules as in filter conjugations. These results indicate that the nodule creates conditions for gene transfer that are comparable to optimal laboratory conditions.     

Alterations in plasma-membrane functions after poliovirus infection

After exposure of HeLa cells to poliovirus there is a rapid decline (within minutes) in fluorescence polarization of DPH (1,6-diphenyl-1,3,5-hexatriene). Within one hour after infection the (Na+/K+)ATPase activity of an isolated plasma-membrane-rich fraction is enhanced, the cell volume decreases, and the intracellular concentration of a potent low-molecular-weight inhibitor of host protein synthesis increases.     

Effect of dietary phosphate intake on phosphate transport by isolated rat renal brush-border vesicles

Renal brush-border membrane vesicles isolated from rats kept for 6-8 weeks on a low-phosphate diet (0.15% of dry matter) showed a markedly faster Na(+)-dependent phosphate uptake than did membrane vesicles isolated from animals kept on a high-phosphate diet (2% of dry matter). Phosphate-uptake rate by brush-border membrane vesicles isolated from animals on a low-phosphate diet remained significantly increased after acute parathyroidectomy. Dietary adaptation was also observed in animals that had been parathyroidectomized before exposure to the different diets. In animals on the low-phosphate diet parathyrin administration inhibited phosphate uptake by brush-border vesicles only if the animals were repleted with P(i) (5ml of 20mm-NaH(2)PO(4)) 1h before being killed. After acute phosphate loading and parathyrin administration the difference in the transport rate between the two dietary groups remained statistically significant. The results suggest that the adaptation of proximal-tubule phosphate transport to dietary intake of phosphate is reflected in the Na(+)/phosphate co-transport system located in the luminal membrane of the proximal-tubule cell. Since the dietary effects on phosphate transport by brush-border membranes are only partially reversed by acute changes in parathyrin concentration and are also observed in chronically parathyroidectomized animals, the adaptation of the Na(+)/phosphate co-transport system to dietary phosphate intake seems to involve an additional mechanism independent of parathyrin.