Improved Microtechnique for the Leptospiral Microscopic Agglutination Test

A method for improving the original Galton microtechnique for detecting leptospiral antibodies has been developed. Simultaneous titrations were performed on 281 animal and human sera and 17 hyperimmune sera with the microscopic agglutination (MA) test and the improved microtechnique. Reproducibility of the improved microtechnique was determined independently on 65 animal sera by two laboratory sections. The results obtained by comparing positive test data from human and animal sera indicated that agreement between the original MA test and this new method exceeded 94%, whereas the original Galton microtechnique and the original MA test agreed in a maximum of 77% of the tests. This study indicates that the results obtained with the improved microtechnique are much more comparable to results obtained with the original MA test than are those obtained with the original Galton microtechnique.     

Cross-linking reconsidered: binding and cross-linking fields and the cellular response.

We analyze a model for the reversible cross-linking of cell surface receptors by a collection of bivalent ligands with different affinities for the receptor as would be found in a polyclonal anti-receptor serum. We assume that the amount of cross-linking determines, via a monotonic function, the rate at which cells become activated and divide. In addition to the density of receptors on the cell surface, two quantities, the binding field and the cross-linking field, are needed to characterize the cross-linking curve, i.e., the equilibrium concentration of cross-linked receptors plotted as a function of the total ligand site concentration. The binding field is the sum of all ligand site concentrations weighted by their respective binding affinities, and the cross-linking field is the sum of all ligand site concentrations weighted by the product of their respective binding and cross-linking affinity and the total receptor density. Assuming that the cross-linking affinity decreases if the binding affinity decreases, we find that the height of the cross-linking curve decreases, its width narrows, and its center shifts to higher ligand site concentrations as the affinities decrease. Moreover, when we consider cross-linking-induced proliferation, we find that there is a minimum cross-linking affinity that must be surpassed before a clone can expand. We also show that under many circumstances a polyclonal antiserum would be more likely than a monoclonal antibody to lead to cross-linking-induced proliferation.     

Neuronal pigmented autophagic vacuoles: lipofuscin, neuromelanin, and ceroid as macroautophagic responses during aging and disease

The most striking morphologic change in neurons during normal aging is the accumulation of autophagic vacuoles filled with lipofuscin or neuromelanin pigments. These organelles are similar to those containing the ceroid pigments associated with neurologic disorders, particularly in diseases caused by lysosomal dysfunction. The pigments arise from incompletely degraded proteins and lipids principally derived from the breakdown of mitochondria or products of oxidized catecholamines. Pigmented autophagic vacuoles may eventually occupy a major portion of the neuronal cell body volume because of resistance of the pigments to lysosomal degradation and/or inadequate fusion of the vacuoles with lysosomes. Although the formation of autophagic vacuoles via macroautophagy protects the neuron from cellular stress, accumulation of pigmented autophagic vacuoles may eventually interfere with normal degradative pathways and endocytic/secretory tasks such as appropriate response to growth factors.     

Quantifying aggregation of IgE-FcepsilonRI by multivalent antigen

Aggregation of cell surface receptors by multivalent ligand can trigger a variety of cellular responses. A well-studied receptor that responds to aggregation is the high affinity receptor for IgE (FcepsilonRI), which is responsible for initiating allergic reactions. To quantify antigen-induced aggregation of IgE-FcepsilonRI complexes, we have developed a method based on multiparameter flow cytometry to monitor both occupancy of surface IgE combining sites and association of antigen with the cell surface. The number of bound IgE combining sites in excess of the number of bound antigens, the number of bridges between receptors, provides a quantitative measure of IgE-FcepsilonRI aggregation. We demonstrate our method by using it to study the equilibrium binding of a haptenated fluorescent protein, 2,4-dinitrophenol-coupled B-phycoerythrin (DNP25-PE), to fluorescein isothiocyanate-labeled anti-DNP IgE on the surface of rat basophilic leukemia cells. The results, which we analyze with the aid of a mathematical model, indicate how IgE-FcepsilonRI aggregation depends on the total concentrations of DNP25-PE and surface IgE. As expected, we find that maximal aggregation occurs at an optimal antigen concentration. We also find that aggregation varies qualitatively with the total concentration of surface IgE as predicted by an earlier theoretical analysis.     

The absolute concentration of nigral neuromelanin, assayed by a new sensitive method, increases throughout the life and is dramatically decreased in Parkinson’s disease

The concentration of neuromelanin (NM) in substantia nigra pars compacta (SNPC) has been measured in male and female normal subjects at different ages in the range 1–97 years old and in SNPC of parkinsonian patients. A very similar age trend of NM concentration was found in both sexes. In the first year of life NM was not detectable, between 10 and 20 years the NM levels were 0.3–0.8 μg/mg of SNPC, between 20 and 50 years were 0.8–2.3 μg/mg SNPC and between 50 and 90 were 2.3–3.7 μg/mg of SNPC. In parkinsonian subjects, the NM levels were 1.2–1.5 μg/mg of SNPC, which is less than 50% with respect to the age-matched controls. These data demonstrate a continuous NM accumulation in SNPC neurons during aging, the presence of large amounts of NM in SNPC and severe depletion of NM in Parkinson’s disease.     

Serial propagation of Pneumocystis carinii in cell line cultures.

Pneumocystis carinii was propagated on three cell lines routinely cultured in many laboratories; the method is practical and convenient. Organisms produced were found to be reactive to Pneumocystis antisera. Studies of antigenic relationships, life cycles, and diagnostic methods will be made easier by these cultures.     

Effects of Wild-Type and Mutated Copper/Zinc Superoxide Dismutase on Neuronal Survival and l-DOPA-Induced Toxicity in Postnatal Midbrain Culture

Abstract: Mutations in the free radical-scavenging enzyme copper/zinc superoxide dismutase (Cu/Zn-SOD) are associated with neuronal death in humans and mice. Here, we examine the effects of human wild-type (WT SOD) and mutant (Gly⁹³→ Ala; G93A) Cu/Zn-SOD enzyme on the fate of postnatal midbrain neurons. One-week-old cultures from transgenic mice expressing WT SOD enzyme had significantly more midbrain neurons and fewer necrotic and apoptotic neurons than non-transgenic cultures. In contrast, 1-week-old cultures from transgenic G93A mice expressing mutant SOD enzyme had significantly fewer midbrain neurons and more necrotic and apoptotic neurons than nontransgenic cultures. To subject postnatal midbrain neurons to oxidative stress, cultures were incubated with l -DOPA. l -DOPA at 200 µ M caused ∼50% loss of tyrosine hydroxylase (TH)-positive neurons in nontransgenic cultures and even greater loss in transgenic G93A cultures; no alterations were noted in GABA neuron numbers. In contrast, 200 µ M l -DOPA did not cause any significant reductions in TH-positive or GABA neuron numbers in transgenic WT SOD cultures. l -DOPA at 50 µ M had opposite effects, in that it significantly increased TH-positive, but not GABA neuron numbers in transgenic WT SOD and G93A and in nontransgenic cultures. These results indicate that increased amounts of WT SOD enzyme promote cell survival and protect against l -DOPA-induced dopaminergic neurotoxicity, whereas increased amounts of mutated Cu/Zn-SOD enzyme have inverse effects. As the spontaneous loss and l -DOPA-induced loss of postnatal dopaminergic midbrain neurons appear to be mediated by free radicals, our study supports the view that mutated Cu/Zn-SOD enzyme kills cells by oxidative stress.     

Dynamic exchange between stabilized conformations predicted for hyaluronan tetrasaccharides: comparison of molecular dynamics simulations with available NMR data

Studies of the hyaluronan (HA) tetrasaccharides are important for understanding hydrogen-bonding in the HA polymer, as they are probably the smallest oligomers in which characteristics of the constituent monosaccharides and the polymer are simultaneously exhibited. Here we present extensive molecular dynamics simulations of the two tetrasaccharides of HA in dilute aqueous solution. These simulations have confirmed the existence of intramolecular hydrogen-bonds between the neighboring sugar residues of HA in solution, as proposed by Scott (1989). However, our simulations predict that these intramolecular hydrogen-bonds are not static as previously proposed, but are in constant dynamic exchange on the sub-nanosecond time-scale. This process results in discrete internal motion of the HA tetrasaccharides where they rapidly move between low energy conformations. Specific interactions between water and intramolecular hydrogen-bonds involving the hydroxymethyl group were found to result in differing conformations and dynamics for the two alternative tetrasaccharides of HA. This new observation suggests that this residue may play a key role in the entropy and stability of HA in solution, allowing it to stay soluble up to high concentration. The vicinal coupling constants3 J NHCH of the acetamido groups have been calculated from our aqueous simulations of HA. We found that high values of 3J NHCH approximately 8 Hz, as experimentally measured for HA, are consistent with mixtures of both trans and cis conformations, and thus3 J NHCH cannot be used to imply a purely trans conformation of the acetamido. The rapid exchange of intramolecular hydrogen-bonds indicates that although the structure is at any moment stabilized by these hydrogen-bonds, no one hydrogen-bond exists for an extended period of time. This could explain why NMR often fails to provide evidence for intramolecular hydrogen-bonds in HA and other aqueous carbohydrate structures.