Chlorine resistance of poliovirus isolants recovered from drinking water

Poliovirus 1 isolants were recovered from finished drinking water produced by a modern, well-operated water treatment plant. These waters contained free chlorine residuals in excess of 1 mg/liter. The chlorine inactivation of purified high-titer preparations of two such isolants was compared with the inactivation behavior of two stock strains of poliovirus 1, LSc and Mahoney. The surviving fraction of virus derived from the two natural isolants was shown to be orders of magnitude greater than that of the standard strains. These results raise the question whether indirect drinking water standards based on free chlorine residuals are adequate public health measures, or whether direct standards based on virus determinations might be necessary.     

Hierarchical down-modulation of hemopoietic growth factor receptors

Granulocytes and macrophages can be produced in vitro when progenitor cells from mouse bone marrow are stimulated by any of four distinct colony stimulating factors, Multi-CSF (IL-3), GM-CSF, G-CSF, and M-CSF (CSF-1). At 0 degrees C the four CSFs do not cross-compete for binding to bone marrow cells, indicating that each has a specific cell surface receptor. However, at 21 degrees C or 37 degrees C, Multi-CSF inhibits binding of the other three CSFs and GM-CSF inhibits binding of G-CSF and M-CSF. Rather than competing directly for receptor binding, the binding of Multi-CSF, GM-CSF, or G-CSF to their own receptor induces the down-modulation (and thus activation) of other CSF receptors at 37 degrees C. The pattern and potency of down-modulation activity exhibited by each type of CSF parallels the pattern and potency of its biological activity. We propose a model in which the biological interactions of the four CSFs are explained by their ability to down-modulate and activate lineage-specific receptors.     

Effect of Quinolinic Acid in the Nucleus Basalis Magnocellularis on Cortical High-Affinity Choline Uptake

A transient 45% increase in cortical high-affinity choline uptake (HACU) was observed after an injection of quinolinic acid (QUIN) into the nucleus basalis magnocellularis (nbM) of the rat. This was followed by a steady decline in choline uptake, which resulted in a 46% decrease by day 7. Specific [3H]hemicholinium-3 binding to coronal brain sections showed a similar pattern following injections of QUIN into the nbM. The increase in cortical HACU elicited by QUIN appeared to be dose dependent.     

An A-ELISA to detect hepatitis A virus in estuarine samples.

An amplified enzyme-linked immunosorbent assay (A-ELISA) for detecting and quantifying hepatitis A virus in estuarine water samples is described. The test was five times more sensitive than a standard ELISA and at least two times more sensitive than radioimmunoassay. Test sensitivity was unaffected by the procedures used to concentrate the virus in estuarine samples or by the presence of humic and tannic acids in test samples. Nonspecific reactions were not encountered with a number of enteroviruses or with a rotavirus. A high sensitivity and specificity combined with speed, low cost, and freedom from radiolabels made the A-ELISA useful for detecting hepatitis A virus in environmental samples. The virus was detected in 3 of 20 estuarine water samples examined by A-ELISA.     

Polyethylene glycol precipitation for recovery of pathogenic viruses, including hepatitis A virus and human rotavirus, from oyster, water, and sediment samples.

Polyethylene glycol 6000 precipitation was found to be an effective concentration method that enhanced the chances for detecting human virus pathogens in environmental samples. Percent recoveries from eluates of fresh and estuarine waters with 8% polyethylene glycol 6000 averaged 86 for hepatitis A virus, 77 for human rotavirus Wa, 87 for simian rotavirus SA11, and 68 for poliovirus. Percent recoveries of 97, 40, 97 and 105, respectively, for the same viruses were obtained from oyster eluates by the same procedure. Percent recoveries of 97 for hepatitis A virus and 78 for human rotavirus Wa were obtained from sediment eluates containing 2 M NaNO3 with a final concentration of 15% polyethylene glycol 6000. The polyethylene glycol method was shown to be more effective than the organic flocculation method for recovery of hepatitis A virus and rotaviruses Wa and SA11, but not of poliovirus 1 in laboratory studies. In field trials, hepatitis A virus or rotavirus or both were recovered from 12 of 18 eluates by polyethylene glycol, compared with recovery from 9 of 18 eluates by organic flocculation from fresh and estuarine waters subject to pollution.     

Purification and properties of the catalytic domain of human 3-hydroxy-3-methylglutaryl-CoA reductase expressed in Escherichia coli

Three fragments of the cDNA encoding human 3-hydroxy-3-methylglutaryl-CoA reductase, all incorporating the majority of the catalytic domain of the protein, were subcloned into Escherichia coli expression vectors containing the pL promoter. The two larger expressed fragments (58 and 52 kDa) were soluble and had enzymatic activity, while the smallest (48 kDa) was insoluble. The two active fragments were purified by a combination of conventional techniques and affinity chromatography. A number of properties of the two enzymes were compared including specific activity, kinetic parameters, relative solubility, and cold lability. The 52-kDa enzyme was observed to change from a dimeric to monomeric form and to lose activity at 4 degrees C. In contrast, the 58-kDa enzyme was found to be much less cold labile, and was dimeric at both 20 and 4 degrees C. In order to resolve the number of subunits required to form an active site, the number of inhibitor binding sites for a known inhibitor was determined to be one per subunit in the 58-kDa enzyme.     

Development of a method for concentration of rotavirus and its application to recovery of rotaviruses from estuarine waters.

As part of our studies on the ecology of human enteric viruses, an improved method for detection of rotaviruses in water was developed, and their presence in Galveston Bay was monitored. Samples (378 liters) of estuarine water adjusted to pH 3.5 and a final AlCl3 molarity of 0.001 were filtered through 25-cm pleated cartridge-type filters (Filterite Corp., Timonium, Md.) of 3.0- and 0.45-micron porosity. Adsorbed virus was eluted with 1 liter of 10% tryptose phosphate broth, pH 9.5. Primary eluates were reconcentrated to a final volume of 10 to 20 ml by a simple and rapid magnetic iron oxide adsorption and elution procedure. Two percent casein at pH 8.5 effectively eluted rotavirus from iron oxide. A total of 21 of 72 samples of water, suspended solids, fluffy sediments, and compact sediments collected in different seasons in Galveston Bay yielded rotaviruses. Recovery of rotaviruses varied from 119 to 1,000 PFU/378 liters of water, 1,200 PFU/1,000 g of compact sediment, 800 to 3,800 PFU/378 liters of fluffy sediment, and 1,800 to 4,980 PFU from suspended solids derived from 378 liters of water based on immunofluorescent foci counts on cover slip cultures of fetal monkey kidney cells.     

Independent regulation of granulocyte-macrophage colony-stimulating factor and multi-lineage colony-stimulating factor production in T lymphocyte clones

When murine T lymphocyte clones were cultured with purified recombinant IL 2, a dose-dependent increase in the production of granulocyte-macrophage colony-stimulating factor (GM-CSF) was observed. Whereas these clones produced both GM-CSF and multi-lineage CSF (multi-CSF) when cultured with concanavalin A, IL 2 induced the production of GM-CSF in the virtual absence of detectable multi-CSF. In addition, IL 2 synergistically enhanced the production of both GM-CSF and multi-CSF by some antigen- or Con-A-stimulated clones. Like Con-A-induced CSF production, GM-CSF production in the presence of IL 2 required protein synthesis but could occur in the absence of proliferation by the clone. Analysis of dose-response curves for stimulation of CSF production by Con A in the presence and absence of IL 2 suggested that Con A and IL 2 activated GM-CSF synthesis by different mechanisms. These results indicate that the coordinate production of two factors by a single T cell clone stimulated with Con A can be dissociated when the clone is stimulated with IL 2.     

Monoclonal antibodies directed against protoplasts of soybean cells: analysis of the lateral mobility of plasma membrane-bound antibody MVS- 1

A monoclonal antibody (MVS-1) was used to monitor the lateral mobility of a defined component (Mr approximately 400,000) of the plasma membrane of soybean protoplasts prepared from suspension cultures of Glycine max (SB-1 cell line). The diffusion coefficient (D) of antibody MVS-1 bound to its target was determined (D = 3.2 X 10(-10) cm2/s) by fluorescence redistribution after photobleaching. Pretreatment of the protoplasts with soybean agglutinin (SBA) resulted in a 10-fold reduction of the lateral mobility of antibody MVS-1 (D = 4.1 X 10(-11) cm2/s). This lectin-induced modulation could be partially reversed by prior treatment of the protoplasts with either colchicine or cytochalasin B. When used together, these drugs completely reversed the modulation effect induced by SBA. These results have refined our previous analysis of the effect of SBA on receptor mobility to the level of a defined receptor and suggest that the binding of SBA to the plasma membrane results in alterations in the plasma membrane such that the lateral diffusion of other receptors is restricted. These effects are most likely mediated by the cytoskeletal components of the plant cell.     

Persistently infected cultures as a source of hepatitis A virus.

Primary African green monkey kidney, continuous African green monkey kidney cell line BS-C-1, and buffalo green monkey kidney cultures were infected with a uniform inoculum of hepatitis A virus (HAV). Although both the cell line BS-C-1 and primary African green monkey kidney cultures produced useful amounts of virus, HAV was detected earlier and in greater quantities in primary African green monkey kidney cultures. A persistently infected primary African green monkey kidney culture was developed. The influence of incubation time (4 to 40 days) and concentration (2 to 15%) of fetal calf serum in the maintenance medium on production of HAV by this culture was examined. An incubation period of 24 to 28 days was found to be optimal; reducing this period led to decreased yields of HAV. No significant difference in the amount of HAV produced was observed with differing concentrations of fetal calf serum. Three different methods of extraction and the effect of multiple extractions on the recovery of HAV from cell lysates were examined. Sonication was a critical factor. Two extractions yielded more than 90% recoverable virus. Yields in excess of 10(11) physical particles of HAV per 850-cm2 roller bottle were routine. The total yield could be increased by concentrating the HAV present in spent maintenance medium by using bentonite or organic flocculation.