Changing patterns of localization of the tobacco mosaic virus movement protein and replicase to the endoplasmic reticulum and microtubules during infection

Tobacco mosaic virus (TMV) derivatives that encode movement protein (MP) as a fusion to the green fluorescent protein (MP:GFP) were used in combination with antibody staining to identify host cell components to which MP and replicase accumulate in cells of infected Nicotiana benthamiana leaves and in infected BY-2 protoplasts. MP:GFP and replicase colocalized to the endoplasmic reticulum (ER; especially the cortical ER) and were present in large, irregularly shaped, ER-derived structures that may represent "viral factories." The ER-derived structures required an intact cytoskeleton, and microtubules appeared to redistribute MP:GFP from these sites during late stages of infection. In leaves, MP:GFP accumulated in plasmodesmata, whereas in protoplasts, the MP:GFP was targeted to distinct, punctate sites near the plasma membrane. Treating protoplasts with cytochalasin D and brefeldin A at the time of inoculation prevented the accumulation of MP:GFP at these sites. It is proposed that the punctate sites anchor the cortical ER to plasma membrane and are related to sites at which plasmodesmata form in walled cells. Hairlike structures containing MP:GFP appeared on the surface of some of the infected protoplasts and are reminiscent of similar structures induced by other plant viruses. We present a model that postulates the role of the ER and cytoskeleton in targeting the MP and viral ribonucleoprotein from sites of virus synthesis to the plasmodesmata through which infection is spread.     

Bacterial hemagglutination by Neisseria gonorrhoeae

Direct bacterial hemagglutination was investigated with 20 clinical isolates of Neisseria gonorrhoeae. The hemagglutination tests were performed by both a macrotechnique with glass slides and a microtechnique with autotrays. Only organisms from form type 1 or 2 colonies caused hemagglutination. There was no statistical difference at a 10% or higher level in hemagglutination powers of type 1 and type 2 organisms, of male urethral and female cervical isolates, and of the eight major human blood types (ABO-Rh). Of seven erythrocyte species tested, only human cells were agglutinated. D-Mannose did not prevent the agglutination. Rabbit antigonococcal serum and high-titer antigonococcal human sera inhibited the hemagglutination. The results suggest the pili are the mediators of hemagglutination and that their specific agglutination of human erythrocytes may be a correlate of their adherence to human mucosal cells in natural infection. Also, although the procedure is presently insensitive, it is possible to detect human antigonococcal antibody by inhibition of direct bacterial hemagglutination.     

The role of state policies and programs in buffering the effects of poverty on children's immunization receipt

OBJECTIVES: This study assessed the influence of public policies on the immunization status of 2-year old children in the United States. METHODS: Up-to-dateness for the primary immunization series was assessed in a national sample of 8100 children from the 1988 National Maternal and Infant Health Survey and its 1991 Longitudinal Follow-Up. RESULTS: Documented immunization rates of this sample were 33% for poor children and 44% for others. More widespread Medicated coverage was associated with greater likelihood of up-to-dateness among poor children. Up-to-dateness was more likely for poor children with public rather than private sources of routine pediatric care, but all children living in states where most immunizations were delivered in the public sector were less likely to be up to date. Poor children in state with partial vaccine replacement programs were less likely to be up to date than those in free-market purchase states. CONCLUSIONS: While state policies can enhance immunization delivery for poor children, heavy reliance on public sector immunization does not ensure timely receipt of vaccines. Public- and private-sector collaboration is necessary to protect children from vaccine-preventable diseases.     

Proteolysis of native proteins. Trapping of a reaction intermediate

When limited proteolysis of the mouse major urinary proteins by trypsin was stopped by rapid denaturation of the proteinase, a covalent adduct of the two proteins was observed. The formation of this complex required active trypsin, was favored at low pH, and could be reversed by the addition of covalent or non-covalent trypsin inhibitors. Electrospray mass spectrometry of the complex demonstrated that it was an acyl-enzyme complex, formed after an unusual exopeptidase attack on the C-terminal-Arg-Glu-OH sequence by trypsin. The complex could sequester over 50% of the trypsin in a digestion mixture, and as anticipated, the protein was an effective trypsin inhibitor.     

Molecular fine-specificity analysis of antibody responses to human cytomegalovirus and design of novel synthetic-peptide-based serodiagnostic assays

BACKGROUND: Glomerular monocyte infiltration is an early feature of lipid-mediated renal injury in animal models. Interactions between mesangial and infiltrating mononuclear cells may contribute to the development of glomerular scarring. METHODS: Adherence of U-937 monocytes to low-density lipoprotein (LDL)- or tumor necrosis factor alpha (TNFalpha)-prestimulated human mesangial cells was assessed by colorimetry of nuclear staining with crystal violet. Blocking antibodies were added to examine the mechanisms of binding. Adhesion molecule expression and fibronectin synthesis were measured by ELISA. RESULTS: Preincubation of mesangial cells for 24 hours with LDL (100 micrograms/ml) or mildly oxidized (minimally modified) LDL (MM-LDL) increased monocyte adhesion by 207% and 240%, respectively, compared with control nonstimulated cells (100%). TNFalpha (100 U/ml) enhanced binding by 335% and up-regulated intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) expression by 505% and 179%, respectively, as compared with MM-LDL (120% and 116%) and LDL, which had no effect. Blocking antibodies to these adhesion molecules inhibited monocyte binding to TNFalpha- and, to a lesser extent, MM-LDL-primed mesangial cells, but had no effect after LDL pretreatment. In contrast to TNFalpha, MM-LDL and LDL increased mesangial cell-associated fibronectin, whereas antibodies to fibronectin inhibited monocyte binding to lipoprotein-stimulated but not TNFalpha-stimulated cells. CONCLUSIONS: Although enhanced monocyte adhesion to TNFalpha- and, to a lesser extent, MM-LDL-stimulated mesangial cells is mediated by changes in ICAM-1 and VCAM-1 expression, both LDL and MM-LDL promote similar cellular interactions as a result of increased fibronectin production.