Low-density lipoprotein preparation by combined diafiltration and ultracentrifugation

A method for isolating low-density lipoprotein by combining diafiltration and ultracentrifugation is described. Diafiltration separates plasma components by use of an ultrafiltration membrane that excludes particles of molecular weight greater than 300,000. The retentate is concentrated three- to fourfold by ultrafiltration, allowing large-scale preparation of low-density lipoprotein. Low-density lipoprotein prepared in this manner is similar in physical, chemical, and biologic properties to low-density lipoprotein isolated by sequential density ultracentrifugation alone. When low-density lipoprotein, prepared by either method, was added to human umbilical vein endothelial cell cultures, no cytotoxicity was observed. The techniques described reduce the demand on multiple rotors and ultracentrifuges for large-scale preparation of low-density lipoprotein suitable and often needed for tissue culture studies.     

Maximization of Candida utilis cytochromes by pulse additions of ethanol to continuous cultures

Candida utilis was grown with glucose as growth-limiting carbon source in batch culture, steady-state continuous culture, and non-steady-state continuous culture. High cytochrome concentrations were routinely recorded in cells harvested in the latter stages of batch culture. They were occasionally recorded in cells from imprecisely controlled steady-state cultures, but precise control of the steady-state dissolved oxygen tension stabilized the cytochromes at relatively low levels. Controlled non-steady-state continuous cultures, imposed by pulse additions of ethanol, routinely produced cells with high cytochrome concentrations. A mechanism is proposed whereby maintenance of cytochrome derepression in continuous culture is dependent upon indefinitely prolonging an “overshoot” response in gene expression.     

Spinoreticular neurons that receive group III input are inhibited by MLR stimulation.

In decerebrate paralyzed cats, we examined the responses of 18 spinoreticular neurons to electrical stimulation of the mesencephalic locomotor region. The activity of each of the spinoreticular neurons was recorded extracellularly from laminae IV through VI of the L(7) and S(1) spinal cord. In addition, each of the 18 spinoreticular neurons received group III afferent input from the tibial nerve. Spinoreticular projections were established for each of 18 neurons by antidromic invasion of the ventro lateral medulla at the P11 though P14 levels. The onset latencies and current thresholds for antidromic invasion from the ventro lateral medulla averaged 15.0 +/- 3.8 ms and 117 +/- 11 microA, respectively. Electrical stimulation of the mesencephalic locomotor region attenuated the spontaneous activity or the responses of each of the spinoreticular neurons to tibial nerve stimulation at currents that recruited group III afferents. Our data support the notion that thin-fiber muscle afferent input to the ventrolateral medulla is gated by a central command to exercise.     

Ntau-methylhistidine excretion is a valid index of myofibrillar protein breakdown in the guineapig (Cavia porcellus).

The recovery of radioactivity in the urine of guineapigs following a bolus intravenous dose of chromatographically pure 14C-Ntau-methylhistidine was measured in order to test whether the excretion of Ntau-methylhistidine (Ntau-MH) is a valid index of myofibrillar protein breakdown in these animals. Four male and four female guineapigs were dosed and after 7 days, 91.65+/-2.82% and 3.58+/-0.91% of injected radioactivity was recovered in the excreta and tissues, respectively. The average total recovery of 95.2+/-3.0% was not significantly different from 100%. Male guineapigs excreted the radioactivity more slowly than females (70% of the dose excreted within 74 h vs 39 h, respectively) but cumulative excretion at 7 days was the same for each sex. Chromatographic analysis of the urine showed almost all of the radioactivity to be associated with a single peak corresponding to Ntau-MH, indicating a lack of significant metabolism. These data show that although the clearance of 14C-Ntau-MH is slower than in rats or humans the urinary excretion of Ntau-MH is a valid index for myofibrillar protein degradation in the guineapig.     

In vitro replication of mouse hepatitis virus strain A59.

An in vitro replication system for mouse hepatitis virus (MHV) strain A59 was developed using lysolecithin to produce cell extracts. In extracts of MHV-infected cells, radiolabeled UMP was incorporated at a linear rate for up to 1 h into RNA, which hybridized to MHV-specific cDNA probes and migrated in denaturing formaldehyde-agarose gels to the same position as MHV genomic RNA. The incorporation of [32P]UMP into genome-sized RNA in vitro correlated with the observed increase of [3H]uridine incorporation in MHV-infected cells labeled in vivo. Incorporation of [32P]UMP into genome-sized RNA was inhibited when extracts were incubated with puromycin. The addition to the assay of antiserum to the MHV-A59 nucleocapsid protein N inhibited synthesis of genome-sized RNA by 90% compared with the addition of preimmune serum. In contrast, antiserum to the E1 or E2 glycoproteins did not significantly inhibit RNA replication. In vitro-synthesized RNA banded in cesium chloride gradients as a ribonucleoprotein complex with the characteristic density of MHV nucleocapsids isolated from virions. These experiments suggest that ongoing protein synthesis is necessary for replication of MHV genomic RNA and indicate that the N protein plays an important role in MHV replication.     

Cloning in Escherichia coli of genes involved in the synthesis of proline and leucine in Desulfovibrio desulfuricans Norway

Summary A library of Deusulfovibrio desulfuricans Norway genomic DNA was constructed in Escherichia coli with pBR322 as vector and plasmids able to complement the proA and leuB mutations of the host were screened. It was observed that all the plasmids studied were highly unstable, the insert DNA being rapidely lost under non-selective growth conditions. A 2.75 kb DNA fragment of D. desulfuricans Norway was found to complement E. coli ProA, ProB and ProC deficiencies. From the results of restriction analysis and Southern hybridization, it is proposed that the genes involved in proline and leucine biosynthesis are clustered on the chromosome of D. desulfuricans Norway.